Suzanne de Bruijn

194 Chapter 4 For the PPCs, total RNA was extracted using a Nucleospin RNA kit (Machery-Nagel) and cDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad). qPCR analysis was performed using GoTaq qPCR Master Mix (Promega) following manufacturer’s instructions. 100 day old ROs were harvested and RNA was extracted using RNeasy Mini Kits (Qiagen). cDNA was synthesized using Tetro cDNA Synthesis kits (Bioline) and qPCR analysis was performed using the SYBR Green labTAQ Green mix (labTAQ) following manufacturer’s instructions on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Primers were designed to assess differential expression of genes implicated in the SVs, and control reference genes and retinal progenitor genes ( Table S3 ). Primers to detect retinal enhancer expression were designed based on observed transcriptional activity of the enhancer RNA in the FANTOM5 Cap Analysis of Gene Expression (CAGE) human dataset ( Table S3 ). 28 Relative gene expression levels, compared to the reference genes GUSB and ACTB , were determined with the ΔΔCt method. 15 Statistical analyses were performed using an unpaired Student t-test to test for significance between groups. SUPPLEMENTARY RESULTS Refinement of the RP17-locus in two unrelated adRP families Index family NL1; In total, 35 affected and 28 unaffected individuals were included. Assuming complete penetrance of the phenotype, a refined locus of 5.16 Mb was identified; chr17:g.55,112,092-60,271,924 (rs8078110-rs9910672) ( Figure 1D ), with a maximumLOD-score of 15.0. Next,WESwas performed in three affected familymembers fromdifferent branches of the family. No rare coding or splice site heterozygous variants (MAF ≤0.0001) located within the defined locus were identified that were shared by all three individuals. In addition, no rare shared heterozygous variants were found in IRD-associated genes (RetNet). Subsequently, WGS was performed in three additional affected individuals. Shared variants within the locus between the three affected individuals were prioritized based on population frequency (MAF ≤0.0001), and coding, splice site, intronic and intergenic heterozygous variants were assessed ( Table S4 ). Index family UK1; WES was performed for an affected individual from a genetically unexplained UK adRP family (UK1). No rare coding or splice site heterozygous variants (MAF ≤0.0001) were identified in IRD-associated genes, so WGS was performed for four affected individuals ( Figure 1B ). Prioritization of rare heterozygous variants in genome data shared by affected individuals in this family failed to identify a candidate rare shared heterozygous variant in IRD-associated genes (MAF ≤0.0001); however, a disease