RSPH3 Mutations Cause Primary Ciliary Dyskinesia with Central-Complex Defects and a Near Absence of Radial Spokes

Abstract

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive condition resulting from structural and/or functional defects of the axoneme in motile cilia and sperm flagella. The great majority of mutations identified so far involve genes whose defects result in dynein-arm anomalies. By contrast, PCD due to CC/RS defects (those in the central complex [CC] and radial spokes [RSs]), which might be difficult to diagnose, remains mostly unexplained. We identified non-ambiguous RSPH3 mutations in 5 of 48 independent families affected by CC/RS defects. RSPH3, whose ortholog in the flagellated alga Chlamydomonas reinhardtii encodes a RS-stalk protein, is mainly expressed in respiratory and testicular cells. Its protein product, which localizes within the cilia of respiratory epithelial cells, was undetectable in airway cells from an individual with RSPH3 mutations and in whom RSPH23 (a RS-neck protein) and RSPH1 and RSPH4A (RS-head proteins) were found to be still present within cilia. In the case of RSPH3 mutations, high-speed-videomicroscopy analyses revealed the coexistence of immotile cilia and motile cilia with movements of reduced amplitude. A striking feature of the ultrastructural phenotype associated with RSPH3 mutations is the near absence of detectable RSs in all cilia in combination with a variable proportion of cilia with CC defects. Overall, this study shows that RSPH3 mutations contribute to disease in more than 10% of PCD-affected individuals with CC/RS defects, thereby allowing an accurate diagnosis to be made in such cases. It also unveils the key role of RSPH3 in the proper building of RSs and the CC in humans.

Radial Spokes—A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

“…However, these sperm swim rapidly with a 9þ0 flagellum of a high beat frequency. Importantly, the typical waveform is helical, similar to the movement of the respiratory cilia of RS PCD patients (e.g., Castleman et al 2009;Jeanson et al 2015) and reactivated axonemes of paralyzed Chlamydomonas RS or CP mutants under altered conditions (e.g., Yagi and Kamiya 2000;and see Loreng and Smith 2016).…”

“…Its velocity is reduced, attributed to altered waveform, likely because of the deficit of inner dynein arms (Vasudevan et al 2015) and uncoordinated beat as expected of minor RS deficits. Unexpectedly, cilia of human RS PCD patients beat rhythmically with helical waveform-amid asynchronous beating ones in some cases-despite lacking spoke heads in RS1 and RS2 or most of RSs (Burgoyne et al 2014;Jeanson et al 2015). The helical movement is attributed to the presence of RS3, because Chlamydomonas flagella with same genetic defect and lacking RS3 are immotile.…”

Value of transmission electron microscopy for primary ciliary dyskinesia diagnosis in the era of molecular medicine: Genetic defects with normal and non-diagnostic ciliary ultrastructure

“…45,46 Mutations in RSPH4A and RSPH3 result in approximately 50% of cilia with normal ultrastructure, while the remainder have varying defects, including roughly 20% missing the central apparatus altogether and 10% with “8+1” ciliary translocation arrangements (one peripheral microtubule shifted to the center to compensate for the missing central pair). 45,47,48 [Figure 7] Detailed imaging studies of transposition defects with TEM and electron tomography explain this varied pattern as a twisting of the ciliary axoneme, resulting in a normal central apparatus (9+2 arrangement) at the ciliary base, with loss of the central pair in the mid-section of the axoneme (9+0 arrangement), and reappearance of a transposed peripheral doublet to the ciliary center (8+1 arrangement) in the distal portion of the axoneme. 49 [Figure 8] Finally, mutations in RSPH1 result in overall normal TEM images, but meticulous inspection reveals only 80% of the axonemes are normal, with the remaining 20% showing various anomalies, such as absence of the central pair or translocation of an outer doublet to the center.…”

“…51 Mutations in RSPH3 also produce abnormal immunofluorescent staining of RSPH3 protein. 47 All radial spoke head gene mutations show abnormal HSVA, mostly with an abnormal rotational beat pattern when viewed from above. However, RSPH1 mutations may result in sections of ciliary biopsies with completely normal ciliary beat frequency and pattern.…”

“…11 Mutations in RSPH3 account for at least 10% of PCD cases with central apparatus/radial spoke defects. 47 The prevalence of the RSPH4A gene is increased in the Irish Traveler, United Kingdom Pakistani, and Puerto Rican populations. 38,45,48 Mutations in radial spoke head genes are detected on most commercial PCD genetic panels.…”