Computational protocol: Novel NR2F1 variants likely disrupt DNA binding: molecular modeling in two cases, review of published cases, genotype–phenotype correlation, and phenotypic expansion of the Bosch–Boonstra–Schaaf optic atrophy syndrome

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Protocol publication

[…] Whole-exome sequencing (WES) was performed on genomic DNA extracted from all samples submitted. The exome was captured utilizing a custom reagent developed by the Mayo Clinic and Agilent Technologies, targeting 19,456 genes and 187,715 exons using 637,923 probes to capture a 54.1-Mbp total region. Sequencing was performed on an Illumina HiSeq 2500 Next-Generation sequencing instrument, using HapMap Sample NA12878 as an internal control. Paired-end 101-bp reads were aligned to a modified human reference genome (GRCh37/hg19) using Novoalign (Novocraft Technologies). Sequencing quality was evaluated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All germline variants were jointly called through GATK Haplotype Caller and GenotypeGVCF (). Each variant was annotated using the BioR Toolkit () and subsequently evaluated for clinical relevance. Sequence coverage is detailed in . [...] We generated a molecular model of the DNA-bound zinc-finger domain of NR2F1 using the homologous (54% identical) crystal structure of RXRA (1BY4 []) and validated it against the experimentally solved solution structure (DOI: 10.2210/pdb2ebl/pdb.). Explicit solvent molecular dynamics (MD) simulations were carried out using NAMD () and the CHARMM27 with CMAP () force field. The wt system was solvated (), ions added to 150 mM NaCl, and computational mutagenesis used to make the C86F model. The wt and C86F proteins were minimized, heated, and equilibrated over a combined 5 nsec each, and in duplicate (NPT). A further 25 nsec of simulation trajectory was generated for each (NVT) and the final 20 nsec analyzed. Additional and independent duplicate 2 nsec explicit solvent MD simulations were generated using the CHARMM force field within Discovery Studio (). Simulations of wt and C86F were also performed without DNA present (apo) and at two temperatures: 300°K and 360°K. Triplicate simulations of each condition were performed in NAMD with systems prepared similarly to above. For each, 22 nsec of simulation was generated and the final 15 nsec analyzed. Thus, a total of 384 nsec (264 nsec apo, 120 nsec holo) of MD simulation was leveraged to characterize the effects of C86F.We additionally studied four previously reported pathogenic variants: R112K, R142L, F110del, and C128R. Triplicate explicit solvent MD simulations of these four variants, wt, and C86F were generated by a similar procedure to the above but with the following modifications: First, the bound DNA fragment was restrained in simulation to limit its dynamics. Second, the protein was initially constrained using harmonic restraints and these restraints were slowly released over 5 nsec. Finally, 20 nsec of production simulation was generated and analyzed.Analysis was carried out using custom scripts, leveraging VMD () and the Bio3D R package (). Protein structure visualization was performed in PyMol (“The PyMOL Molecular Graphics System. Version 1.5.0.3”) and VMD. Prior to analysis, all trajectories were aligned to the initial wt conformation using Cα atoms. PC analysis was performed using Cα atoms in Cartesian space. […]

Pipeline specifications

Software tools NAMD, VMD, PyMOL
Application Protein structure analysis
Organisms Homo sapiens