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. 2015 Oct;53(4):563-73.
doi: 10.1165/rcmb.2014-0483OC.

Immunofluorescence Analysis and Diagnosis of Primary Ciliary Dyskinesia with Radial Spoke Defects

Adrien Frommer  1 , Rim Hjeij  1 , Niki T Loges  1 , Christine Edelbusch  1 , Charlotte Jahnke  1 , Johanna Raidt  1 , Claudius Werner  1 , Julia Wallmeier  1 , Jörg Große-Onnebrink  1 , Heike Olbrich  1 , Sandra Cindrić  1 , Martine Jaspers  2 , Mieke Boon  3 , Yasin Memari  4 , Richard Durbin  4 , Anja Kolb-Kokocinski  4 , Sascha Sauer  5 , June K Marthin  6 , Kim G Nielsen  6 , Israel Amirav  7 , Nael Elias  8 , Eitan Kerem  9 , David Shoseyov  9 , Karsten Haeffner  10 , Heymut Omran  1
Affiliations

Immunofluorescence Analysis and Diagnosis of Primary Ciliary Dyskinesia with Radial Spoke Defects

Adrien Frommer et al. Am J Respir Cell Mol Biol. 2015 Oct.

Abstract

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder caused by several distinct defects in genes responsible for ciliary beating, leading to defective mucociliary clearance often associated with randomization of left/right body asymmetry. Individuals with PCD caused by defective radial spoke (RS) heads are difficult to diagnose owing to lack of gross ultrastructural defects and absence of situs inversus. Thus far, most mutations identified in human radial spoke genes (RSPH) are loss-of-function mutations, and missense variants have been rarely described. We studied the consequences of different RSPH9, RSPH4A, and RSPH1 mutations on the assembly of the RS complex to improve diagnostics in PCD. We report 21 individuals with PCD (16 families) with biallelic mutations in RSPH9, RSPH4A, and RSPH1, including seven novel mutations comprising missense variants, and performed high-resolution immunofluorescence analysis of human respiratory cilia. Missense variants are frequent genetic defects in PCD with RS defects. Absence of RSPH4A due to mutations in RSPH4A results in deficient axonemal assembly of the RS head components RSPH1 and RSPH9. RSPH1 mutant cilia, lacking RSPH1, fail to assemble RSPH9, whereas RSPH9 mutations result in axonemal absence of RSPH9, but do not affect the assembly of the other head proteins, RSPH1 and RSPH4A. Interestingly, our results were identical in individuals carrying loss-of-function mutations, missense variants, or one amino acid deletion. Immunofluorescence analysis can improve diagnosis of PCD in patients with loss-of-function mutations as well as missense variants. RSPH4A is the core protein of the RS head.

Keywords: cilia; human radial spoke protein 1; human radial spoke protein 4A; human radial spoke protein 9; primary ciliary dyskinesia.

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Figures

Figure 1.
Figure 1.
Model of a ciliary cross-section and the radial spoke (RS) complex based on immunofluorescence (IF) findings and Chlamydomonas orthology. Ciliary cross-section showing the typical 9 + 2 ultrastructure (A) of nine outer microtubulus doublets arranged around the central pair. Outer dynein arms, inner dynein arms, and RSs emerge from each A-microtubule. As illustrated, the RS complexes connect the outer microtubulus doublets to the central pair. The RS proteins, RSPH9, RSPH4A, and RSPH1, are located in the head complex and arranged in a twofold rotational symmetry, whereas RSPH23 is found in the neck and RSPH3 in the stalk compartment. The location of these proteins is indicated by the numbers 9, 1, 4A, 23, 3 (B). Model of the RS complex was created based on published findings in Chlamydomonas reinhardtii (, , , , –49).
Figure 2.
Figure 2.
Western blot analyses of antibodies directed against human radial spoke (RSPH) proteins. The antibodies used in this study, directed against RSPH9 (A), RSPH4A (B), RSPH1 (C), RSPH23 (D), and RSPH3 (E), were validated by Western blot and specifically detect bands with the predicted molecular weight or similar to the predicted molecular weight of the proteins.
Figure 3.
Figure 3.
Loss-of-function mutations in the human radial spoke genes RSPH9, RSPH4A, and RSPH1 disrupt the assembly of the RS head protein, RSPH9. Respiratory epithelial cells from control subjects (A) and individuals affected by primary ciliary dyskinesia (PCD) carrying biallelic loss-of-function mutations in RSPH9 (OP-1135 [B]), RSPH4A (OI-162 [C]), or RSPH1 (OP-1428 II1 [D]) were double labeled with antibodies directed against acetylated α-tubulin (green) and RSPH9 (red). Both proteins colocalize (yellow) along the ciliary axonemes in cells from the unaffected control subjects (A). In contrast, in respiratory cilia of individuals with loss-of-function mutations in RSPH9, RSPH4A, and RSPH1, RSPH9 is undetectable in ciliary axonemes (B–D), indicating that the ciliary localization of RSPH9 is RSPH4A and RSPH1 dependent. Nuclei were stained with Hoechst 33342 (blue). acet. Tubulin, acetylated α-tubulin; DIC, differential interference contrast. Scale bars, 10 μm.
Figure 4.
Figure 4.
Assembly of the RS head protein, RSPH4A, into ciliary axonemes is affected by loss-of-function mutations in RSPH4A but not by mutations in RSPH1 and/or RSPH9. Respiratory epithelial cells from control (A) and individuals affected by PCD carrying biallelic loss-of-function mutations in RSPH9 (OP-1135 [B]), RSPH4A (OI-162 [C]), or RSPH1 (OP-1428 II1 [D]) were double labeled with antibodies directed against acetylated α-tubulin (green) and RSPH4A (red). Both proteins colocalize (yellow) along the ciliary axonemes in cells from the unaffected control subjects and RSPH9 or RSPH1 mutant individuals (A, B, and D). In contrast, in respiratory cilia of RSPH4A mutant individuals, RSPH4A is undetectable in the ciliary axonemes (C), indicating that the ciliary localization of RSPH4A is affected by mutations in RSPH4A but not by mutations in RSPH9 (B) or RSPH1 (D). Nuclei were stained with Hoechst 33342 (blue). Scale bars, 10 μm.
Figure 5.
Figure 5.
RSPH4A and RSPH1 loss-of-function mutations disrupt the assembly of the RS head protein, RSPH1, whereas mutations in RSPH9 do not. Respiratory epithelial cells from control subjects (A) and individuals affected by PCD carrying biallelic loss-of-function mutations in RSPH9 (OP-1135 [B]), RSPH4A (OI-162 [C]), or RSPH1 (OP-1428 II1 [D]) were double labeled with antibodies directed against acetylated α-tubulin (green) and RSPH1 (red). Both proteins colocalize (yellow) along the ciliary axonemes in cells from the unaffected control subjects and the RSPH9 mutant individual (A and B). In contrast, in respiratory cilia of individuals with loss-of-function mutations in RSPH4A or RSPH1, RSPH1 is undetectable in the ciliary axonemes (C and D), indicating that the ciliary localization of RSPH1 is also affected by loss-of-function mutations in RSPH4A but not in RSPH9. Nuclei were stained with Hoechst 33342 (blue). Scale bars, 10 μm.
Figure 6.
Figure 6.
Summary of the defects caused by mutations in RSPH9, RSPH4A, and RSPH1. Compared with wild-type (WT) RSs (A), mutation of RSPH9 solely leads to deficiency of the RSPH9 projection (B). Based on our IF findings, we conclude that deficiency of the RSPH1 projection leads to the lack of RSPH9 and RSPH1 (C), whereas RSPH4A mutants lack the entire head complex, just leaving the neck and stalk unaffected (D). Therefore, we conclude the following ladder of importance for the correct assembly of the RS head, starting with the most significant: RSPH4A, RSPH1, and RSPH9.
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