Fig. 4

Complex NODAL indel variants in two probands with congenital heart disease. A Pedigree structure for Family 6 (top) with integrative genomics viewer (IGV) view of the heterozygous complex indel variant in NODAL (below). Under the IGV view are Sanger dideoxy sequence traces for the proband, LAT0191 (left) and mother, LAT0192, (right). The Sanger sequence trace panels at top represent PCR amplification products of the variant region from genomic DNA. Individual alleles are not discernable for the proband genomic DNA trace at left. Beneath the genomic DNA results are Sanger sequence traces from cloning the amplified variant region using TA cloning kits as described in “Methods”. Top and bottom panels are representative of different populations of clones for each allele. At left, the complex c.700_723delinsTTGACTTCC, p.R234_P241delinsLTS variant is apparent in the Allele 2 (Mut) Sanger trace for the proband, LAT0191. B Pedigree, IGV view, and Sanger traces for Family 7. In addition to the c.700_723delins, p.R234_P241delinsLTS variant, Sanger traces for the nearby c.778G > A:p.G260R variant are shown. Allele 1 in LAT1763 is shown to be WT for the c.700_723 locus, while containing the c.778G > A variant. Allele 2 was shown to contain the c.700_723delins, p.R234_P241delinsLTS variant while being WT at the c.778G > A locus, confirming these variant alleles are in a trans configuration. C, D Secondary structure predictions for WT and mutant RNA using the RNAfold Server (http://rna.tbi.univie.ac.at/) using the minimum free energy (C) and thermodynamic ensemble (D) functions. Base pair probability is shown using color coding; cooler colors (blue) represent lower probability and warmer colors (red) represent higher probability. Variant RNA has more stable secondary structure as shown at right in C and D. E UCSC genome browser view of the 12 clones sequenced for LAT0191 and LAT1763, showing populations with the deletion, and with WT sequence. All variant data shown is for NODAL transcript NM_018055.5