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. 2015 Jul 2;97(1):153-62.
doi: 10.1016/j.ajhg.2015.05.004. Epub 2015 Jun 11.

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

Ludovic Jeanson  1 , Bruno Copin  2 , Jean-François Papon  3 , Florence Dastot-Le Moal  2 , Philippe Duquesnoy  1 , Guy Montantin  2 , Jacques Cadranel  4 , Harriet Corvol  5 , André Coste  6 , Julie Désir  7 , Anissa Souayah  8 , Esther Kott  1 , Nathalie Collot  2 , Sylvie Tissier  2 , Bruno Louis  9 , Aline Tamalet  10 , Jacques de Blic  11 , Annick Clement  12 , Estelle Escudier  13 , Serge Amselem  14 , Marie Legendre  13
Affiliations

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

Ludovic Jeanson et al. Am J Hum Genet. .

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.

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Figures

Figure 1
Figure 1
CC/RS Defects in Respiratory Cilia of Individuals with RSPH3 Mutations (A) The electron micrographs of cross-sections of cilia from a control individual and five individuals with identified RSPH3 mutations are shown. For each affected individual, two sections are shown: one with a well-organized configuration (9+2) showing the presence of the CC but with a near absence of RSs (top) and another with an abnormal axonemal configuration (9+2 disorganized or 8+1 transposition) and no detectable RSs (bottom). Black scale bars represent 0.1 μm. (B) Distribution of the different ciliary defects (expressed as a percentage of abnormal cilia) identified by TEM in individuals with RSPH3 mutations. Reference values are those from the TEM study performed by Rossman et al. on 55 control (non-atopic non-smoker healthy) individuals: the proportion of cilia with a normal ultrastructure is 95.2% ± 3.5%, whereas that of cilia with a missing CC is 0.4% ± 0.8%. Abbreviations are as follows: IDA, inner dynein arms; ODA, outer dynein arms.
Figure 2
Figure 2
RSPH3 Mutations and Their Impact at the Protein Level in Individuals with PCD Exonic organization of the human RSPH3 cDNA containing the mutations for the five families described in this study (top). Domain-organization models of the corresponding protein (middle) and of the Chlamydomonas reinhardtii orthologous RSP3 (bottom) are also shown. The eight exons are indicated by empty or hashed boxes, which depict translated or untranslated sequences, respectively. According to the predictions by SMART and literature, RSPH3 is composed of a radial spoke 3 domain (RS3D, in red) containing an axoneme targeting domain (ATD, in yellow), a RIIa-domain-binding amphipathic helix (AHR, in blue), a Dpy-30-domain-binding amphipathic helix (AHD, in blue), three TQT-like LC8-binding motifs (T, in purple), two coil-coiled domains (CC, in green), and two phosphorylated threonines (P, orange circle).
Figure 3
Figure 3
Localization of RSPH3 and Other Ciliary Proteins in Airway Epithelial Cells from a Healthy Individual and Individual PCD9 RSPH3 (green) localized within cilia (red) of the airway epithelial cells of a healthy control (A–C) and was absent from the cilia of individual PCD9 (A′–C′). RSPH23 (D–F and D′–F′), RSPH1 (G–I and G′–I′), and RSPH4A (J–L and J′–L′) (all in green) localized within cilia (red) of the airway epithelial cells of a healthy control individual and of individual PCD9. RSPH11 (green) localized within cilia (red) of the airway epithelial cells of a healthy control individual (M–O) but was absent from the cilia of individual PCD9 (M′–O′). DNALI1 (P–R and P′–R′) (in green) localized within cilia (red) of the airway epithelial cells of a healthy control individual and of individual PCD9. Airway epithelial cells were examined after labeling with rabbit polyclonal antibodies directed against RSPH3 (Novus Biological NBP1-84244, 1:100, 37°C, 1 hr), RSPH23 (Sigma HPA044555, 1:200, 37°C, 1 hr), RSPH1 (Sigma HPA017382, 1:200, 37°C, 1 hr), RSPH4A (Sigma HPA031196, 1:200, 37°C, 1 hr), RSPH11 (Sigma HPA039193, 1:200, 37°C, 1 hr), or DNALI1 (Abcam ab87075, 1:400, 37°C, 1 hr) and a secondary goat anti-rabbit Alexa Fluor 488 (green) antibody (Invitrogen A11034). For control individuals, we used an antibody directed against acetylated α-tubulin (mouse monoclonal [6-11B-1], Abcam ab24610, 1:700) to visualize microtubules, revealed by a secondary goat anti-mouse Alexa Fluor 594 (red) antibody (Invitrogen A11032). Nuclei were stained with DAPI (Sigma 32670). White scale bars represent 10 μm.
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