Apping peaks at the indicated position inside the box. In (A) a cytosine is substituted by a thymine, in (B) an adenine is substituted by a cysteine, in (C) a guanine is substituted by a thymine, and in (D) a cytosine is substituted by a thymine. doi:10.1371/journal.pone.0049532.gNFATC1 and Tricuspid Atresiaincubation for 20 minutes the samples were loaded and the gel was run for 2.5 hours. The gel was then dried using the BioRad gel dryer (Model 583) for 2 hours at 80uC followed by exposition to a PhosphoImager screen. The results were visualized using the STORM scanner (General Electric). Quantification of the bands was done using TotalLab2010 (General Electric).Statistical AnalysisThe significance of the luciferase transcriptional assays was analyzed using the one-way Anova single test (p,0.05).BioinformaticsThe NFATC1 secondary structures were predicted and visualized by the Discovery Studio program (Acclerys Inc.). Briefly, the human NFATC1 protein sequence was imported from the SWISS database and the secondary amino acid structure was predicted based on the nature and structure of the composing amino acids using already validated approaches by the Discovery Studio. The mutated amino acids were introduced to the same sequence, and the prediction of the structure was carried on using the same approach.Figure 2. Mendelian inheritance of the different NFATC1 SNPs. Genotype-phenotype correlations showed that in addition to the indexed patient with tricuspid atresia who died at 17 years of age, his “healthy” father carried the four different SNPs. None of the siblings, nor the mother who all are healthy have any of these SNPs. doi:10.1371/journal.pone.0049532.gThe buy HIF-2��-IN-1 nuclear extracts were run on a 6 non-denaturing polyacrylamide gel (Acrylamide: Bis (29:1), 1.6 APS, TEMED, water and 0.25X TBE) in 0.25X TBE buffer at 200 volts. The reaction consisted of 10 mg of extracts, 4 ml binding buffer (20 mM Tris pH 7.9, 120 mM KCl, 2 mM EDTA, 25 mM MgCl2 and 25 glycerol), 1 ml poly dI/dC (General Electric) and 1 ml of the probe. The reaction was completed to 20 ml with water. AfterResultsPatients with various heart valve defects were recruited as part of the ongoing study on the genetics of CHD in the Lebanese population. The subjects’ list included a total of 135 patients and 100 control healthy individuals. 23727046 The distribution of valvular CHDs among patients was as follows: tricuspid atresia (19 patients), pulmonary stenosis (63 patients, 9 of which are strictly valvular),Figure 3. Effects of the two missense SNPs on the structure of the NFATC1 protein. A- The missense SNPs lead to a P66L substitution at the N-terminal region of the protein near the calcineurin-docking site (Cln binding), and to a I701L substitution at the C-terminal region downstream of the Rel Homolgy Domain (RHD). The schematic represents isoform A, the most abundant NFATC1 protein with 717 amino acids, a transactivation domain (TAD) at the N-terminus, and a DNA binding domain 
at the C-terminus. (NLS = nuclear localization signal, NES = nuclear export signal, and SP = Serine-Proline). B- The NFATC1 secondary structures were predicted and visualized by the Discovery Studio program (Acclerys Inc.). The results demonstrate the formation of a new ML 281 site beta-sheet in the P66L mutant and a deletion of a beta-sheet in the I701L mutant as compared to the wild type protein. doi:10.1371/journal.pone.0049532.gNFATC1 and Tricuspid AtresiaTable 2. Frequency of the NFATC1 mutations according to t.Apping peaks at the indicated position inside the box. In (A) a cytosine is substituted by a thymine, in (B) an adenine is substituted by a cysteine, in (C) a guanine is substituted by a thymine, and in (D) a cytosine is substituted by a thymine. doi:10.1371/journal.pone.0049532.gNFATC1 and Tricuspid Atresiaincubation for 20 minutes the samples were loaded and the gel was run for 2.5 hours. The gel was then dried using the BioRad gel dryer (Model 583) for 2 hours at 80uC followed by exposition to a PhosphoImager screen. The results were visualized using the STORM scanner (General Electric). Quantification of the bands was done using TotalLab2010 (General Electric).Statistical AnalysisThe significance of the luciferase transcriptional assays was analyzed using the one-way Anova single test (p,0.05).BioinformaticsThe NFATC1 secondary structures were predicted and visualized by the Discovery Studio program (Acclerys Inc.). Briefly, the human NFATC1 protein sequence was imported from the SWISS database and the secondary amino 
acid structure was predicted based on the nature and structure of the composing amino acids using already validated approaches by the Discovery Studio. The mutated amino acids were introduced to the same sequence, and the prediction of the structure was carried on using the same approach.Figure 2. Mendelian inheritance of the different NFATC1 SNPs. Genotype-phenotype correlations showed that in addition to the indexed patient with tricuspid atresia who died at 17 years of age, his “healthy” father carried the four different SNPs. None of the siblings, nor the mother who all are healthy have any of these SNPs. doi:10.1371/journal.pone.0049532.gThe nuclear extracts were run on a 6 non-denaturing polyacrylamide gel (Acrylamide: Bis (29:1), 1.6 APS, TEMED, water and 0.25X TBE) in 0.25X TBE buffer at 200 volts. The reaction consisted of 10 mg of extracts, 4 ml binding buffer (20 mM Tris pH 7.9, 120 mM KCl, 2 mM EDTA, 25 mM MgCl2 and 25 glycerol), 1 ml poly dI/dC (General Electric) and 1 ml of the probe. The reaction was completed to 20 ml with water. AfterResultsPatients with various heart valve defects were recruited as part of the ongoing study on the genetics of CHD in the Lebanese population. The subjects’ list included a total of 135 patients and 100 control healthy individuals. 23727046 The distribution of valvular CHDs among patients was as follows: tricuspid atresia (19 patients), pulmonary stenosis (63 patients, 9 of which are strictly valvular),Figure 3. Effects of the two missense SNPs on the structure of the NFATC1 protein. A- The missense SNPs lead to a P66L substitution at the N-terminal region of the protein near the calcineurin-docking site (Cln binding), and to a I701L substitution at the C-terminal region downstream of the Rel Homolgy Domain (RHD). The schematic represents isoform A, the most abundant NFATC1 protein with 717 amino acids, a transactivation domain (TAD) at the N-terminus, and a DNA binding domain at the C-terminus. (NLS = nuclear localization signal, NES = nuclear export signal, and SP = Serine-Proline). B- The NFATC1 secondary structures were predicted and visualized by the Discovery Studio program (Acclerys Inc.). The results demonstrate the formation of a new beta-sheet in the P66L mutant and a deletion of a beta-sheet in the I701L mutant as compared to the wild type protein. doi:10.1371/journal.pone.0049532.gNFATC1 and Tricuspid AtresiaTable 2. Frequency of the NFATC1 mutations according to t.
