Computational protocol: Autosomal dominant transmission of complicated hereditary spastic paraplegia due to a dominant negative mutation of KIF1A, SPG30 gene

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Protocol publication

[…] The study included 4 subjects from a non-consanguineous family from Korea presenting with cognitive impairment, spastic gait, and negative test result for the spastic paraplegia 4 (SPG4) gene. Written informed consent was obtained from the family for the blood samples and the study was approved by the Institutional Review Board (IRB) of the Pusan National University Yangsan Hospital (IRB number: 05–20117–052). All experiments in this study were performed in accordance with the relevance guidelines. Library preparation was performed with a TruSight One Sequencing Panel Kit (Illumina, USA). This panel enriches for approximately 4,800 genes of clinical relevance. Massive parallel sequencing was conducted with a NextSeq. 500 sequencing instrument (Illumina, USA). Local realignment and recalibration were performed using the Genome Analysis Tool Kit (GATK version 3.30). Among the 7,853 called variants, 1,215 remained after filtering for common variants with a minor allele frequency (1%) using multiple population databases (1000 Genomes Project, Exome Variant Server, and Exome Aggregation Consortium). Nucleotide numbering of the mutations herein reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the NCBI reference sequence NM_001244008.1, while the amino positions are based on the corresponding NCBI reference sequence NP_001230937.1. Prediction of the effects of the de novo missense KIF1A mutations on the protein function were done using PolyPhen-2 ( and SIFT (, and MutationTaster (, respectively. [...] Structural modeling of the KIF1A motor with p.Thr258Met mutation was performed using the crystal structure of the KIF1A motor domain in complex with ADP [PDB ID: 1I5S], which has a complete structure of L11, unlike other reported crystal structures of KIF1A with unmodelled L11 structure for the lack of electron density in this region. The p.Thr258Met point mutation was introduced to the KIF1A motor domain using the mutagenesis option in PyMOL, a molecular visualization software. Energy minimization and loop flexible modeling were performed using Modeller software. All structural images were generated using PyMOL. […]

Pipeline specifications

Software tools PyMOL, MODELLER
Application Protein structure analysis