Computational protocol: Functional characterization of a GFAP variant of uncertain significance in an Alexander disease case within the setting of an individualized medicine clinic

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Protocol publication

[…] The monomer sequence was annotated according to the presence of known autosomal dominant variants (http://www.waisman.wisc.edu/alexander-disease), variants indexed by the Intermediate Filament Database , pathogenic variants from ClinVar , and HGMD . The domain architecture used was according to Uniprot (P14136) . Paralog annotation analysis was performed using a previously described method . Briefly, for this purpose multiple sequence alignment (MSA) of paralogs was generated using paralog sequences gathered from Ensembl gene ENSG00000131095 using annotations from GRCh37.p13. The MSA was constructed using Clustal Omega , with default parameters. Published variants in paralogs were obtained from PubMed and projected on the sequence of human GFAP. Protein secondary structure was predicted using PsiPred . Homology‐based protein structure models were constructed using Modeller , version 9.15. The tetramer was generated from homology‐based modeling using a vimentin tetramer model , as a template. Intra‐ and intermolecular interactions, including salt bridge interactions, hydrogen bonds, electrostatic interactions, and hydrophobic interactions, were calculated in the Receptor‐Ligand function of Discovery Studio Client 4.0 . In silico mutagenesis was performed using the Accelrys Builder software . Molecular models were first energy minimized using a two‐step protocol of steepest descent and conjugated gradients. All these steps were performed using the SHAKE procedure. A distance‐dependent dielectrics implicit solvent model was used with a dielectric constant of 80 and a pH of 7.4 as previously described . […]

Pipeline specifications

Software tools Clustal Omega, PSIPRED, MODELLER
Databases ClinVar HGMD
Application Protein structure analysis
Organisms Homo sapiens
Diseases Alexander Disease