Suzanne de Bruijn

80 Chapter 2 from the Netherlands. This study was approved by the Institutional Review Boards of the participating centers, and adhered to the tenets of the declaration of Helsinki. All subjects provided informed consent prior to inclusion in the study. Clinical data were collected from the medical records of two patients from Family A (A-II:1, A-II:2) and one patient from Family B (B-II:1), including information regarding best-corrected Snellen visual acuity, and results of slit-lamp biomicroscopy and ophthalmoscopy. In patient A-II:2 and B-II:1, fundus photography, spectral-domain optical tomography (SD-OCT; Spectralis, Heidelberg Engineering) and Goldmann kinetic perimetry were performed, and full-field electroretinography was recorded according to the International Society for Clinical Electrophysiology of Vision guidelines and assessed by applying local standard values. 6 In addition, fundus autofluorescence (Spectralis, Heidelberg Engineering) images, were available for patient B-II:1. Whole exome sequencing and variant interpretation Genomic DNA was isolated from peripheral blood using standard isolation methods and WES was performed in both probands. For proband A-II:2, exome enrichment was performed using the Agilent SureSelect Human All Exome V6 kit. Read mapping along the hg19 reference genome (GrCH37/hg19) and variant calling were performed using BWA version 0.78 7 and the haplotype caller module of GATK (Broad Institute). 8 Copy number variant (CNV) detection was performed using CoNIFER version 0.2.2. 9 Exome enrichment for proband B-II:1 was carried out with the Agilent SureSelect XT Human All ExonV5 enrichment kit. Mapping of sequencing reads along the hg19 reference genome and variant calling were performed using Lifescope version 1.3 (Life Technologies). CNV detection was performed using ExomeDepth version 1.1.1. 10 For both datasets, the obtained variants were filtered based on population allele frequencies ≤0.5% in gnomAD 11 , ExAC 12 , dbSNP 13 , and an in-house exome database (containing 15,576 alleles). Only nonsense, indels, splice site (-14/+14 nucleotides), missense and synonymous variants were assessed. Missense variants were only assessed when predicted to be possibly pathogenic by at least one in silico predictor; a Grantham score ≥80, PhyloP ≥2.7 or CADD-Phred score ≥15. 14 Synonymous variants were only assessed when predicted to have an effect on splicing by one of the splice prediction tools that are embedded in the AlamutVisual software (version 2.10). Candidate genes in which remaining variants were found were compared to currently known IRD-associated genes listed on RetNet 3 (accessed on 1 st June 2018). Validation of found variants and segregation analysis were performed by Sanger sequencing. Primer sequences and PCR conditions are available upon request.