Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia

Abstract

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia

“…Mutations in nine genes affect outer dynein arm (ODA) components: DNAI1, DNAI2, DNAH5, DNAL1, CCDC103, NME8/TXNDC3, CCDC114 and ARMC4 [1,6,7] result in ODA defects, whereas DNAH11 mutations result in PCD with normal ultrastructure [8]. Nine genes encode the cytoplasmic proteins required for the assembly of both ODA and inner dynein arm (IDA) complexes: KTU/DNAAF1, LRRC50/DNAAF2, C19orf51/DNAAF3, LRRC6, HEATR2, DYX1C1/DNAAF4, ZMYND10, SPAG1 and C21orf59 [1,[9][10][11][12]. Mutations in CCDC39 and CCDC40 result in an IDA and microtubular disorganisation defect [13,14].…”

Diagnostic Methods in Primary Ciliary Dyskinesia

“…Mutations in CCDC39 [82,83], CCDC40 [82,84], CCDC65 [85] and CCDC164 [63] Numerous proteins are involved in cytoplasmic biogenesis of cilia. The identification of genes involved in cytoplasmic assembly of cilia (HEATR2, DNAAF1, DNAAF2, DNAAF3, DYX1C1, CCDC103, LRRC6, ZMYND10, SPAG1, ARMC4 and C21orf59) has provided clear insights into this process [81,[85][86][87][88][89][90][91][92][93][94][95].…”

“…The identification of genes involved in cytoplasmic assembly of cilia (HEATR2, DNAAF1, DNAAF2, DNAAF3, DYX1C1, CCDC103, LRRC6, ZMYND10, SPAG1, ARMC4 and C21orf59) has provided clear insights into this process [81,[85][86][87][88][89][90][91][92][93][94][95]. Defects in the assembly line can result in ODA or ODA/IDA defects.…”

Sperm dysfunction and ciliopathy

“…From studies in Chlamydomonas, this N-DRC structure is thought potentially important in the regulation of IADs. Recent genetic and ultrastructural studies have identified the components of the N-DRC, its associated proteins such as CCDC39, CCDC40, CCDC65, and DRC1 (CCDC164), and their connections with ciliopathy [163,[167][168][169].…”

“…C21orf59/ FBB18 is a flagellar matrix protein and participates in dynein arm assembly. Loss of C21orf59/FBB18 in humans causes deficiency of both ODAs and IDAs [163].…”