Suzanne de Bruijn

82 Chapter 2 reagent (Thermo Fisher Scientific) according to manufacturer’s protocol. After 24 hours of transfection, cells were serum starved (0.2% FCS) for 48 hours to induce ciliogenesis. To assess the effect of the siRNAs on KIAA1549 expression, RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel), and expression was quantified by qPCR. To evaluate the effect of knockdown of the long transcript on expression of the short transcript, HEK293T cells were used. HEK293T cells express both the long and short transcript abundantly, unlike hTERT-RPE1 cells which only express the long transcript. For immunofluorescence, transfected hTERT-RPE1 cells were fixed with 2% PFA for 20 minutes, and permeabilized using 1% Triton X-100 in PBS for 5 minutes. Subsequently, cells were blocked with 2% bovine serum albumin (BSA) in PBS for 45 minutes. Primary antibodies against the primary cilium (anti-ARL13B; 1:500; cat.# 17711-1AP; ProteinTech) and the ciliary transition zone (anti-RPGRIP1L; 1:500; cat.# SNC039; 6 ) diluted in blocking solution were incubated for 1 hour. After incubation with secondary antibodies in blocking solution for 45 minutes, samples were mounted by VECTASHIELD containing DAPI (Vector Laboratories). Cells were imaged using a Zeiss Axio Imager Z2 fluorescence microscope and a 63x magnification. Percentage of ciliated cells and cilium length were calculated using Fiji Is Just ImageJ (FIJI). 15 Each experiment was performed three independent times. RESULTS Identification of KIAA1549 variants To identify the genetic defect underlying the arRP in two affected siblings of an Iranian consanguineous family (Family A; Figure 1A ), exome sequencing was performed in individual A-II:2. After analysis of the WES data, the frameshift variant c.52del (Hg19:g.138,665,964del; p.(Arg18Alafs*64)) was detected in the candidate RP gene KIAA1549 (Family A). This variant is located in the second largest homozygous region of 13.2 Mb. Presence of the homozygous variant was confirmed and segregation analysis was performed using Sanger sequencing. The variant is absent frompopulation frequency databases gnomAD, ExAC, dbSNP and the in-house database. Moreover, the variant is absent from the Iranome database 16 , which contains whole exome sequencing data of 800 healthy individuals from eight major ethnic groups in Iran. The variant causes a frameshift in exon 1, and is predicted to result in degradation of KIAA1549 mRNA due to nonsense-mediated decay. Previously, a heterozygous variant in CRB1 was reported for Family A in both affected siblings. 17 Analysis of theWES data of patient A-II:2 did not yield additional variants or copy number variants in this gene. Moreover, the c.2816A>G (p.(Asn894Ser)) variant was predicted to be benign by in silico predictions