Mice with a Deletion of <i>Rsph1</i> Exhibit a Low Level of Mucociliary Clearance and Develop a Primary Ciliary Dyskinesia Phenotype

(ENGLISH) Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disease caused by mutations in over 40 different genes. Individuals with PCD caused by mutations in RSPH1 (radial spoke head 1 homolog) have been reported to have a milder phenotype than other individuals with PCD, as evidenced by a lower incidence of neonatal respiratory distress, higher nasal nitric oxide concentrations, and better lung function. To better understand genotype–phenotype relationships in PCD, we have characterized a mutant mouse model with a deletion of Rsph1. Approximately 50% of cilia from Rsph1 / cells appeared normal by transmission EM, whereas the remaining cilia revealed a range of defects, primarily transpositions or a missing central pair. Ciliary beat frequency in Rsph1 / cells was significantly lower than in control cells (20.2 ± 0.8 vs. 25.0 ± 0.9 Hz), and the cilia exhibited an aberrant rotational waveform. Young Rsph1 / animals demonstrated a low rate of mucociliary clearance in the nasopharynx that was reduced to zero by about 1 month of age. Rsph1 / animals accumulated mucus in the nasal cavity but had a lower bacterial burden than animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1 / ). Thus, Rsph1 / mice display a PCD phenotype similar to but less severe than that observed in Dnaic1 / mice, similar to what has been observed in humans. The results suggest that some individuals with PCD may not have a complete loss of mucociliary clearance and further suggest that early diagnosis and intervention may of other causes, genetic modifiers or environmental factors. To further test the hypothesis that RSPH1 mutations result in some amount of MCC and a less severe PCD phenotype, as well as to gain insights into the mechanisms that may be responsible, we have characterized the pathogenesis of disease in a mouse model with a deletion of Rsph1 (24). We also compared the Rsph1 / mice with animals with a deletion of dynein axonemal intermediate chain 1 (Dnaic1 / ) that causes ciliary immotility and a complete lack of effective MCC (25). Our results show that Rsph1 / mice demonstrate significant ciliary activity and a low amount of mucociliary transport at young ages (Days 3–11), but mucociliary transport ceases by Day 30. In addition, the Rsph1 / mice also showed a reduced amount of nasal bacterial colonization at early time points compared with the Dnaic1 / mice. These data support the hypothesis that although mutations in RSPH1 disrupt ciliary function and cause PCD in humans, the residual ciliary activity results in a less severe clinical phenotype. / animals were scored as normal. The most common abnormality observed, occurring in 52% of the abnormal axonemes (60 of 115), was a transposition defect, in which the central pair is missing and one of the outer doublets has migrated into the central region of the axoneme (Figures 1A and 1B). Other ciliary abnormalities observed included a missing central pair (28 of 115 [24%]) and translocations (7 of 115 [6%]) (Figures 1A and 1B). These results are similar to observations in human patients with PCD with mutations in RSPH1 (34). pair. mice showed no evidence of MCC, analysis of younger animals showed a slow but significant rate of MCC in the nasopharynx. We measured MCC by tracking the distance traveled by fluorescent beads deposited at the anterior tip of the nasopharynx. We have previously reported that in this anatomical location in mice, MCC is particularly robust, even in very young animals (31). Our results demonstrate that in the control animals, the rate of MCC increases with age, possibly due to a progressive increase in the number of ciliated cells, as has been observed in the trachea (39). In the Rsph1 / animals, the rate of MCC also appeared to increase over the first 10 days but was markedly reduced compared with the control animals. Between 10 and 11 days of age, the rate of MCC in the PCD animals was approximately 20% of that in the control animals. Thus, although the absence of Rsph1 clearly results in defective ciliary structure, a reduced CBF, and an aberrant circular waveform, young Rsph1 / mice maintain a low but significant amount of MCC, at least in the nasopharynx. This residual amount of MCC was lost over time, suggesting that it was not sufficient to prevent mucus accumulation and further disease progression in the nasal cavity. Because MCC in the Rsph1 / mice is inefficient, it is possible that mucus simply accumulates until the defective Rsph1 / cilia can no longer provide enough force to maintain MCC. It is also possible that the properties of the mucus itself may be different in older animals, owing to developmental changes or in response to environmental challenges. For example, we have observed that the abundance of mucous secretory cells in the trachea and bronchi of neonatal mice is altered in a reproducible developmental pattern between Days 5 and 10 (47). Alternatively, the presence of accumulated mucus may itself trigger a response that alters the composition and properties of airway secretions in the older Rsph1 / animals. Although it is not clear why MCC ceases in the Rsph1 / mouse model, the initial low amount of MCC may explain the less severe phenotype of PCD in patients with RSPH1 mutations. Although mutations in RSPH1 disrupt the axonemal structure of respiratory cilia and alter the beat pattern from planar to