Splice-Site Mutations in the Axonemal Outer Dynein Arm Docking Complex Gene CCDC114 Cause Primary Ciliary Dyskinesia Alexandros Onoufriadis, Tamara Paff,

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Splice-Site Mutations in the Axonemal Outer Dynein Arm Docking Complex Gene CCDC114 Cause Primary Ciliary Dyskinesia Alexandros Onoufriadis, Tamara Paff, Dinu Antony, Amelia Shoemark, Dimitra Micha, Bertus Kuyt, Miriam Schmidts, Stavroula Petridi, Jeanette E. Dankert-Roelse, Eric G. Haarman, Johannes M.A. Daniels, Richard D. Emes, Robert Wilson, Claire Hogg, Peter J. Scambler, Eddie M.K. Chung, Gerard Pals, Hannah M. Mitchison The American Journal of Human Genetics Volume 92, Issue 1, Pages 88-98 (January 2013) DOI: 10.1016/j.ajhg.2012.11.002 Copyright © 2013 The American Society of Human Genetics Terms and Conditions

Figure 1 Segregation Analysis of CCDC114 Mutations (A) Pedigree structure of Volendam families PCD-01–PCD-08 showing the segregation of the c.742G>A mutation and of UK family PCD-09 (boxed) showing segregation of the c.486+1G>A mutation. The genealogy of PCD-01 is derived from available church records. Not all ascertained individuals have been shown in the pedigrees, for reasons of space. Filled symbols indicate affected individuals, clear symbols indicate unaffected individuals, gray indicates affected individuals for whom samples could not be obtained, diamonds and dashed symbols indicate confirmed older individuals where samples are unavailable. Asterisks indicate situs abnormalities were reported. (B) High-resolution computed tomography (HRCT) chest scan of an affected Volendam individual showing bronchiectasis of the right and left lower lobes of the lung. The American Journal of Human Genetics 2013 92, 88-98DOI: (10.1016/j.ajhg.2012.11.002) Copyright © 2013 The American Society of Human Genetics Terms and Conditions

Figure 2 CCDC114 Splice-Site Mutations Causing Primary Ciliary Dyskinesia (A) Effect of the c.742G>A Volendam mutation on splicing. The upper panels show the location of the mutation in genomic DNA sequence chromatograms and the splice-site prediction effect according to Alamut. Alamut uses the four different splice prediction software programs listed on the left. In comparison of the reference sequence from a control individual (top) against the mutant genomic DNA (bottom), the software predicts loss of the splice donor site and presence of a cryptic splice site 79 bp into the intron. The bottom panel shows the sequence of cDNA from a person who is homozygous for the mutation, isolated from ciliary cells and amplified via primers in exons 6 and 8. An intronic insertion of 79 basepairs is present in the c.742G>A individual’s cDNA, located between the mutation substitution site (green arrow) and the presumed intronic cryptic splice site (pink arrow). The sequence shows no indication of use of the regular splice donor site. The inclusion of 79 bases leads to a frameshift and a premature stop codon in exon 8 after addition of 52 novel amino acids, at the in-frame TAA codon indicated by the red box with arrow. (B) Relative expression levels (normalized to ACTB) of CCDC114, CCDC63, and DNAH5 in mRNA from testis and cultured nasal epithelial cells from controls or from Volendam PCD-02 II:2, assessed by qPCR with a Roche Lightcycler as described in Table S5. CCDC114 is expressed at higher levels in cilia-producing cells compared to testis whereas CCDC63 is expressed highly in testis with no detectable expression in cilia-producing cells. In addition, CCDC114 and DNAH5 levels are both reduced in cilia from the Volendam affected individual compared to control. The means ± SEM from triplicate repeat experiments are shown. (C) Location of the Volendam and UK splice-site mutations in the intron-exon structure shown above, and in a model of the CCDC114 protein shown below. Black boxes indicate coding exons, white boxes noncoding exons. The green boxes indicate coiled-coil domains as detected by Paircoil2 run with a minimum window size of 28. Homology was also detected identifying an SMC (structural maintenance of chromosomes protein) domain in CCDC114 indicated by the blue box (SMC_prok_B TIGR02168) and a putative prokaryotic phosphodiesterase domain indicated by the orange box (PRK12704). The American Journal of Human Genetics 2013 92, 88-98DOI: (10.1016/j.ajhg.2012.11.002) Copyright © 2013 The American Society of Human Genetics Terms and Conditions

Figure 3 CCDC114 Splice-Site Mutations Are Associated with Ciliary Axoneme Defects (A) Transmission electron micrographs of cross-sections of respiratory epithelial cell cilia demonstrates loss of outer dynein arms in both the PCD-02 II:2 and PCD-09 II:1 individuals carrying the c.742G>A and c.486+1G>A splice donor mutations, respectively. All nine peripheral doublets showed loss and reduction of the outer dynein arms (arrows) compared to controls. Scale bar represents 100 nm. (B) Subcellular localization of CCDC114 protein (green) in respiratory epithelial cells via a rabbit polyclonal antibody (Sigma HPA042524). In healthy individuals (top), CCDC114 is localized along the length of the axoneme of the ciliated cells, whereas in both PCD-02 II:2 and PCD-09 II:1, CCDC114 is markedly reduced (middle and bottom). Axoneme-specific anti-acetylated-α-tubulin antibody (Sigma) was used as a control to stain the entire axoneme (red). DNA (blue) was stained with DAPI (Invitrogen). Scale bars represent 10 μm. The American Journal of Human Genetics 2013 92, 88-98DOI: (10.1016/j.ajhg.2012.11.002) Copyright © 2013 The American Society of Human Genetics Terms and Conditions