Mutation at a highly conserved arginine residue in the DNA binding domain of <i>NR1D2</i> is associated with AVSD.

<p><b>(a)</b> Multiple alignment of amino acid residues in the C-terminal aspect of the DNA binding domain of NR1D2 show strong conservation through evolution to the <i>Drosophila</i> paralog Eip75B. <b>(b)</b> A 3d representation of the crystal structure of NR1D1 complexed to DNA. The NR1D1 and NR1D2 DNA binding domains display 96% sequence identity (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.s004" target="_blank">S3 Fig</a>), and the altered arginine residue (black arrow) contacts the minor groove of DNA in the C-terminal portion of the DNA binding domain (RCSB 1A6Y) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.ref082" target="_blank">82</a>]. <b>(c)</b> The p.R175W mutation shows increased transcriptional activity in a cell culture assay (<i>p = 0</i>.<i>0022</i>, two-tailed t-test). A wild-type or mutant <i>Nr1d2</i> construct was transfected in HUVEC cells along with a vector containing a synthetic response element driving a GFP reporter. Mean and standard deviation for each condition are displayed. <b>(d)</b> Two representative cardiac lesions in <i>Nr1d2</i>^{<i>tm1-Dgen</i>} <i>-/-</i> mouse hearts. An oblique coronal section of the heart of a spontaneously deceased P0 <i>Nr1d2</i>^{<i>tm1-Dgen</i>} <i>-/-</i> pup reveals a primum ASD and suggests a common AV-valve (white arrow) indicative of AVSD (star). A coronal section of an e17.5 <i>Nr1d2</i>^{<i>tm1-Dgen</i>} <i>-/-</i> heart shows an inlet VSD (plus sign) which is a type of VSD closely related to AVSDs in cardiac development [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.ref078" target="_blank">78</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005963#pgen.1005963.ref083" target="_blank">83</a>]. Abbreviations: LV-left and RV-right ventricles, RA-right atrium, IVS- interventricular septum, VSD-ventricular septal defect, ASD-atrial septal defect.