Computational protocol: Non-syndromic retinitis pigmentosa due to mutations in the mucopolysaccharidosis type IIIC gene, heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT)

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Protocol publication

[…] Genomic DNA was extracted from leukocytes according to standard procedures (). WES of Patient A-II:1 was performed at Otogenetics corporation (Norcross, GA, USA) using Roche NimbleGen V2 (44.1 Mbp) paired-end sample preparation kit and Illumina HiSeq2000 at a 30× coverage. Sequence reads were aligned to the reference human genome (GRCh37/hg19), and variants were called as previously described (). WES of Patients C-II:1, C-II:2 and C-II:3 was performed using Agilent's Sure Select All Human Exome version 2 kit (50 Mb; Agilent Technologies, Santa Clara, CA, USA), followed by sequencing on a SOLiD4 sequencing platform (Life Technologies, Carlsbad, CA, USA). Sequence reads were aligned to the reference human genome (GRCh37/hg19) using Lifescope v2.1 software (Life Technologies), followed by variant calling on the aligned sequence via a customized pipeline ().HGSNAT exons were amplified from genomic DNA using the primers listed in Supplementary material, Table S2. Sanger sequencing of PCR products was used to verify the mutations and screen additional patients and controls.In Patients C-II:1, C-II:2 and C-II:3, copy number variant analysis of HGSNAT exons 3 and 18 was performed on genomic DNA via qPCR using GoTaq qPCR Master Mix (Promega Corporation, Madison, WI, USA) to determine whether a heterozygous deletion was present in HGSNAT. The KEL and ZDHHC5 genes were used as standards (primers sequences in Supplementary material, Table S2). [...] Retnet-Retinal Information Network (http://www.sph.uth.tmc.edu/Retnet/)OMIM (http://www.ncbi.nlm.nih.gov/omim)The 1000 genomes database (http://www.1000genomes.org/)EVS NHLBI Exome Sequencing Project (ESP) Exome Variant Server (http://evs.gs.washington.edu/EVS/)dbSNP (http://www.ncbi.nlm.nih.gov/SNP/)PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)Mutation Taster (http://www.mutationtaster.org/)Analyzer Splice Tool (http://ibis.tau.ac.il/ssat/SpliceSiteFrame.htm)Human Splicing Finder Version 3.0 (http://www.umd.be/HSF3/)Ensembl genome browser (http://www.ensembl.org/index.html)CADD (http://cadd.gs.washington.edu/)Ocular Tissue Database (https://genome.uiowa.edu/otdb/)Human Retinal Transcriptome (http://oculargenomics.meei.harvard.edu/index.php/ret-trans) […]

Pipeline specifications

Software tools LifeScope, PolyPhen, MutationTaster, CADD
Databases dbSNP Exome Variant Server OMIM RetNet
Applications WGS analysis, WES analysis, Genome data visualization
Organisms Mus musculus, Homo sapiens
Diseases Mucopolysaccharidosis III, Nervous System Diseases, Retinal Degeneration, Retinitis Pigmentosa, Genetic Diseases, Inborn