Suzanne de Bruijn

83 Homozygous KIAA1549 variants are associated with retinitis pigmentosa suggesting that CRB1 –variants are unlikely to cause disease in this family. No CNVs or other compound heterozygous or homozygous variants were detected in currently known IRD-associated genes. Also, no heterozygous candidate variants were found in causative genes related to the patient’s phenotype. WES was performed in patient B-II:1 affected with RP (Family B, Figure 1A ), and revealed a homozygous missense variant in KIAA1549 ; c.4686C>A (Hg19:g.138,554,373G>T; p.(His1562Gln)). After analysis, this was the only homozygous variant remaining in an IRD-associated gene and no compound heterozygous variants were observed. The homozygous variant was validated in the proband and segregation analysis was performed by Sanger sequencing in two unaffected siblings. 1 1 2 3 2 1 1 2 3 2 Family A Family B A B M1|M1 M1|M1 M2|M2 M1: c.52del; p.(Arg18Alafs*64) M2: c.4686C>A; p.(His1562Gln) I I II II Homo sapiens Macaca mulatta Rattus norvegicus Mus musculus Canis lupus fam. Gallus gallus Xenopus tropicalis Danio rerio D D D D D D D D E E E E E E E E R R R R R R R R Y Y Y Y Y Y Y Y R R R R R R Q Q Q Q H H H H H H V V V V V V I M T T T T T T S N S K K K K K K R p.(His1562Gln) Q K K R R R R R R R R R R R Q R M2|+ M2|+ Figure 1. KIAA1549 variants detected in patients in two families with retinitis pigmentosa. (A) Pedigrees of two families with RP associated with homozygous variants in KIAA1549 (NM_001164665). (B) Evolutionary conservation of the mutated amino acid (M2) identified in Family B. Black boxes indicate fully conserved amino acid residues, whereas dark grey boxes indicate highly conserved and light grey boxes moderately conserved amino acid residues. M, mutation. The moderately conserved mutated histidine residue is located in a highly conserved region ( Figure 1B ), has a CADD-Phred score of 24.2, a PhyloP score of 0.53 and a Grantham score of 24. Additionally, the prediction tools MutationTaster 18 , PolyPhen-2 19 and SIFT 14 predicted the variant to be disease causing (p-value: 0.835), possibly damaging (HumDiv: 0.889; HumVar: 0.651) and tolerated (0.17), respectively. Moreover, putative changes are predicted by Human Splicing Finder. 20 The binding sites of the splicing factors SRp40 and SF2/ASF are no longer present, and a potential creation of an exonic splicing silencer site is predicted. 20 Heterozygous variants in currently known IRD- associated genes were found in ABCA4 (c.2588G>C(;)5603A>T; p.[Gly863Ala, Gly863del] (;)(Asn1868Ile) and CDHR1 (c.512C>G; p.(Thr171Ser)), but no second pathogenic alleles could be detected for these genes. No CNVs were detected in regions overlapping with known IRD-associated genes.