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. 2013 Sep 5;93(3):561-70.
doi: 10.1016/j.ajhg.2013.07.013. Epub 2013 Aug 29.

Loss-of-function Mutations in RSPH1 Cause Primary Ciliary Dyskinesia With Central-Complex and Radial-Spoke Defects

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Free PMC article

Loss-of-function Mutations in RSPH1 Cause Primary Ciliary Dyskinesia With Central-Complex and Radial-Spoke Defects

Esther Kott et al. Am J Hum Genet. .
Free PMC article

Abstract

Primary ciliary dyskinesia (PCD) is a rare autosomal-recessive respiratory disorder resulting from defects of motile cilia. Various axonemal ultrastructural phenotypes have been observed, including one with so-called central-complex (CC) defects, whose molecular basis remains unexplained in most cases. To identify genes involved in this phenotype, whose diagnosis can be particularly difficult to establish, we combined homozygosity mapping and whole-exome sequencing in a consanguineous individual with CC defects. This identified a nonsense mutation in RSPH1, a gene whose ortholog in Chlamydomonas reinhardtii encodes a radial-spoke (RS)-head protein and is mainly expressed in respiratory and testis cells. Subsequent analyses of RSPH1 identified biallelic mutations in 10 of 48 independent families affected by CC defects. These mutations include splicing defects, as demonstrated by the study of RSPH1 transcripts obtained from airway cells of affected individuals. Wild-type RSPH1 localizes within cilia of airway cells, but we were unable to detect it in an individual with RSPH1 loss-of-function mutations. High-speed-videomicroscopy analyses revealed the coexistence of different ciliary beating patterns-cilia with a normal beat frequency but abnormal motion alongside immotile cilia or cilia with a slowed beat frequency-in each individual. This study shows that this gene is mutated in 20.8% of individuals with CC defects, whose diagnosis could now be improved by molecular screening. RSPH1 mutations thus appear as a major etiology for this PCD phenotype, which in fact includes RS defects, thereby unveiling the importance of RSPH1 in the proper building of CCs and RSs in humans.

Figures

Figure 1
Figure 1
CC and RS Defects in Respiratory Cilia of Individuals with RSPH1 Mutations The electron micrographs of cross-sections of cilia from a control and ten individuals with identified biallelic RSPH1 mutations are shown. For each affected individual, two sections are shown: one with a normal configuration showing the presence of the CC and RSs (top) and the other with an abnormal axonemal configuration characterized by CC and RS abnormalities (bottom). The yellow flashes show the presence of normal CCs in the normal control and the affected individuals; RSs are encircled in yellow in the normal control and in cilia with CCs from affected individuals. The red flashes show CC defects (including the 8+1 pattern in DCP781) in affected individuals. Black scale bars represent 0.1 μm.
Figure 2
Figure 2
RSPH1 Mutations and Their Impact at the Protein Level in Individuals with PCD (A) Exonic organization of the human RSPH1 cDNA, in which are shown the mutations for the ten families described in this study (top), and a domain-organization model of the corresponding protein (middle). The mutations’ impact at the protein level is shown (bottom). The nine exons are indicated by empty or hashed boxes depicting translated or untranslated sequences, respectively. According to the prediction tools provided by the NCBI and UniProt (see Web Resources), RSPH1 contains five MORN repeats, followed by a linker and a sixth MORN repeat. (B) A partial protein alignment of RSPH1 in different species shows the evolutionary conservation of the fourth MORN repeat, which contains the amino acid substitution identified in this study.
Figure 3
Figure 3
RSPH1 Localizes to Cilia and Is Absent from Airway Epithelial Cells of DCP1064 (A–F) In airway epithelial cells of a healthy control (A–C) and the healthy father of DCP1064 (D–F), RSPH1 (green) localizes within cilia (red). (G–I) In cells from individual DCP1064, RSPH1 labeling is absent from cilia. Airway epithelial cells were examined after labeling with a rabbit RSPH1 polyclonal antibody (Sigma HPA017382, 1:100, 37°C, 1 hr) and a secondary goat anti-rabbit Alexa Fluor-488 (green) antibody (Invitrogen, A11034). For controls, we used an antibody directed against acetylated α-tubulin (mouse monoclonal [6-11B-1], Abcam ab24610, 1:700) for the visualization of 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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