Computational protocol: Clinical and Molecular Characteristics of Childhood Onset Stargardt Disease

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Protocol publication

[…] Blood samples were collected in EDTA tubes and DNA was extracted with a Nucleon Genomic DNA extraction kit (BACC2; Tepnel Life Sciences, West Lothian, UK). All 50 exons and exon–intron boundaries of the ABCA4 gene were amplified using Illumina Truseq Custom Amplicon protocol (Illumina, San Diego, CA), followed by sequencing on Illumina MiSeq platform., The next-generation sequencing reads were analyzed and compared with the reference genome GRCh37/hg19, using the variant discovery software NextGENe (SoftGenetics LLC, State College, PA). All detected possibly disease-associated variants were confirmed by Sanger sequencing.,All the missense variants identified were analyzed using 2 software prediction programs: SIFT (Sorting Intolerant from Tolerant; available from; accessed November 1, 2013), and PolyPhen2 (available from; accessed November 1, 2013). Predicted effects on splicing of all the missense and intronic variants were assessed with the Human Splicing finder program version 2.4.1 (available from; accessed November 1, 2013). The allele frequency of all variants was estimated by reference to the Exome Variant Server (NHLBI Exome Sequencing Project, Seattle, WA; available from; accessed November 1, 2013).Patients harboring ≥2 mutations were classified into 3 genotype groups based on mutation type: Group A included patients with ≥2 definitely or likely deleterious (severe) variants; group B included patients with 1 deleterious variant and ≥1 missense or in-frame insertion/deletion variants; and group C included individuals with ≥2 missense or in-frame insertion/deletion variants (). One disease-associated intronic change of unknown effect was treated as a deleterious allele owing to the associated severe clinical phenotype previously reported., It should be noted, however, that assigning severity (e.g., a deleterious effect) to a mutation was not always straightforward, especially for missense alleles and some variants in splice sites. […]

Pipeline specifications

Software tools NextGENe, SIFT, PolyPhen, HSF
Databases Exome Variant Server
Applications WGS analysis, WES analysis
Organisms Homo sapiens
Diseases Retinal Diseases, Cardiovascular Abnormalities