Computational protocol: Determination of genotypic and clinical characteristics of Colombian patients with mucopolysaccharidosis IVA

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Protocol publication

[…] The Ethics Committee of the National University of Colombia approved the study, and 32 patients from different regions of the country were included. According to local regulations, the parents or legal guardians of the minors provided their informed consent for participation before being enrolled (assent of the minors was also obtained). The main inclusion criterion referred to patients diagnosed with MPS IVA via clinical, biochemical and genetic/radiological evaluation to measure the activity of the enzyme N-acetyl-galactosamine-6-sulfate sulfatase in leukocytes. Data was analyzed based on the Review of clinical presentation and diagnosis of mucopolysaccharidosis IVA published in 2013.Exploratory data analysis was performed by using percentages and frequency tables for discrete and categorical variables; continuous variables were analyzed using central tendency and dispersion measures. SPSS (free trial version 21.0) was the statistical software used.Genomic DNA was extracted by using the Ultraclean® Blood DNA Isolation Kit. Amplification of the 14 exons including the intron-exon boundaries of the GALNS gene was carried out with the primers designed employing the online software Primer 3, as reported by Pajares et al and synthesized by Invitrogen (). Therefore, PCR amplification was done in MyCycler and T100 Bio-Rad® thermocyclers. Sequencing was completed by using an ABI PRISM 3500 automated sequencer (Applied Biosystems).For reporting gene variants, retrieved electropherograms were analyzed with program BioEdit v7.2.5 Sequence Alignment Editor (; Tom Hall Ibis Therapeutics (a division of Isis Pharmaceuticals), Carlsbad, CA, USA) and compared to the GALNS reference sequence NG_008667. The new variants were classified and analyzed by using the SIFT platforms (; Craig Venter Institute, CA, USA), PolyPhen 2 (; Biobyte Solutions, Heidelberg, Germany), Mutation Taster (; NCBI 37/Ensembl 69, Schwarz, Cooper, Schuelke, Seelow), PMUT (; IRB Barcelona Institute for Research in Biomedicine), PhD-SNP (, and FATHMM (; University of Bristol Integrative Epidemiology Unit, UK) and taking into account the ACMG recommendations for evaluating the variants.Molecular docking was carried out for wild-type GALNS and mutants. As described by Rivera-Colón et al, homology modeling was performed using the structure of the protein N-acetyl-galactosamine-6-sulfate-sulfatase. Two structures were utilized for this analysis with accession numbers 4FDI and 4FDJ from the Protein Data Bank (PDB) (; Collaborative Research for Structural Bioinformatics: Rutgers and UCSD/SDSC). Modeling was accomplished with a template of wild-type structure of GALNS code 4FDI (due to its 2.2 Å resolution) using the SWISS-model platform. The X, Y, and Z coordinates to be used in AutoDock Tools (version 1.5.6) were calculated with the eFindsite platform (; Louisiana State University). Calculations led to seven options for pocket coordinates, and the authors selected the one with the best confidence interval. Option 1 was chosen for G6S substrate (confidence interval: 0.9580). Molecular docking between the enzyme and the ligand was performed in silico; affinity energy (kcal/mol) values were obtained by using AutoDock Vina 1.0 (; The Scripps Research Institute) and AutoDock Tools (Version 1.5.6) (; The Scripps Research Institute).For estimating the energy values for wild-type GALNS bindings and the mutants models, N-acetylgalactosamine-6-sulfate (GalNAc6S; PubChem CID:193456) was used as the ligand molecular docking results were visualized with LigPlot+ (; EMBL-EBI, Wellcome Genome Campus, Hinxton, Cambridgeshire, UK). […]

Pipeline specifications

Software tools BioEdit, PolyPhen, MutationTaster, PhD-SNP, FATHMM, AutoDock, eFindSite, AutoDock Vina, LigPlot+
Application Protein interaction analysis
Organisms Homo sapiens
Diseases Mucopolysaccharidosis IV, Protein Deficiency