Lack of CFAP54 causes primary ciliary dyskinesia in a mouse model and human patients

Abstract

Primary ciliary dyskinesia (PCD) is a highly heterogeneous recessive inherited disorder. FAP54, the homolog of CFAP54 in Chlamydomonas reinhardtii, was previously demonstrated as the C1d projection of the central microtubule apparatus of flagella. A Cfap54 knockout mouse model was then reported to have PCD-relevant phenotypes. Through whole-exome sequencing, compound heterozygous variants c.2649_2657delinC (p. E883Dfs*47) and c.7312_7313insCGCAGGCTGAATTCTTGG (p. T2438delinsTQAEFLA) in a new suspected PCD-relevant gene, CFAP54, were identified in an individual with PCD. Two missense variants, c.4112A>C (p. E1371A) and c.6559C>T (p. P2187S), in CFAP54 were detected in another unrelated patient. In this study, a minigene assay was conducted on the frameshift mutation showing a reduction in mRNA expression. In addition, a CFAP54 in-frame variant knock-in mouse model was established, which recapitulated the typical symptoms of PCD, including hydrocephalus, infertility, and mucus accumulation in nasal sinuses. Correspondingly, two missense variants were deleterious, with a dramatic reduction in mRNA abundance from bronchial tissue and sperm. The identification of PCD-causing variants of CFAP54 in two unrelated patients with PCD for the first time provides strong supportive evidence that CFAP54 is a new PCD-causing gene. This study further helps expand the disease-associated gene spectrum and improve genetic testing for PCD diagnosis in the future.

Keywords

primary ciliary dyskinesia / CFAP54 / cilia

1 Introduction

Primary ciliary dyskinesia (PCD, OMIM ID: 244400) is a rare, highly heterogeneous, autosomal recessive or X-linked recessive genetic disorder, with an estimated incidence from 1:10 000 to 1:20 000 [1]. The pathogenic mechanism of PCD is associated with the impairment of the structure or function of motile cilia or sperm flagella [2]. Motile cilia are mostly present on the respiratory epithelium, ependymal cells, and ciliated cells in the female reproductive tract and embryo (as nodal cilia, “9+0” axonemal structural cilia without the central microtubule pairs aid the embryo in sensing fluid flow for left-right determination during embryonic development) [3,4]. The malfunction of cilia and flagella leads to neonatal respiratory distress, chronic wet cough, chronic otitis media, sinusitis, bronchiectasis, infertility, and situs inversus [5,6]. To date, more than 40 pathogenic genes of PCD have been reported, most of which refer to axonemal motors, structure and regulation, or ciliary assembly and preassembly [7]. However, some PCD cases remain unsolved, in which the genetic lesions remain unknown.

Virtually, all eukaryotic motile cilia and flagella are highly conserved organelles with a “9+2” structure comprising nine doublet microtubules surrounding a central microtubule pair associated with outer dynein arms, inner dynein arms, nexin-dynein regulatory complexes, radial spokes, and the central apparatus [2,8,9]. The apparatus of the central pair is an asymmetric regulatory complex for axonemal dyneins, and it includes at least seven distinct protein projections: C1a, C1b, C1c, C1d, C2a, C2b, and C2c [10]. A previous mass spectrometry and proteomic study of flagella in Chlamydomonas reinhardtii identified a series of flagella-associated proteins, and among them, flagella-associated protein 54 (FAP54), the ortholog of human CFAP54 protein, was conserved and mainly exists in the axoneme but was functionally uncharacterized [11]. DiPetrillo and Smith’s study demonstrated that FAP54 is a component of the C1d complex, which is associated with calcium-dependent calmodulin binding and regulating the motility of wild-type C. reinhardtii [8,12]. Since then, the function of FAP54 or CFAP54 has been gradually revealed. In 2015, a Cfap54 knockout mouse model was reported to have PCD phenotypes, such as hydrocephalus, defects in spermatogenesis, accumulation of mucus in the sinus cavity, and defects in ciliary structure and function [13]. Three types of Cfap54 mutant mice were generated: one was on a C57BL/6J background with severe hydrocephalus, which resulted in early mortality, and the other two were on a mixed C57BL/6J-129S6/SvEvTac background or 129S6/SvEvTac background with relatively mild hydrocephalus and without signs of early mortality. Moreover, Cfap54 was proven to be mainly expressed in the testis, lung, and brain of mice, further confirming that this gene is relevant to motile cilia. However, CFAP54 has not yet been related to patients with PCD.

This study identified two unrelated patients with PCD, who were born by nonconsanguineous healthy parents, and carrying compound heterozygous mutations in CFAP54 by whole-exome sequencing (WES). In family 1 (F1), the frameshift mutation c.2649_2657delinC (p. E883Dfs*47) was suspected to be pathogenic and may cause a reduction in CFAP54 mRNA expression, which was verified by a minigene assay in vitro. The pathogenicity of the in-frame mutation c.7312_7313insCGCAGGCTGAATTCTTGG (p. T2438delinsTQAEFLA) carried by the patient was investigated in vivo with the use of a knock-in mouse model established by CRISPR/Cas9. The knock-in mouse model consistently presented with PCD phenotypes of hydrocephalus, male infertility, and mucus accumulation in the nasal sinus. In family 2 (F2), the CFAP54 mRNA expression in the bronchial tissue and sperm of individuals with PCD harboring CFAP54 compound missense variants of c.4112A>C (p. E1371A) and c.6559C>T (p. P2187S) significantly decreased. This study was the first to link CFAP54 to PCD cases, and thus, CFAP54 is a new PCD-relevant gene.

2 Materials and methods

2.1 Patient subjects

The proband from F1 is 32 years old diagnosed with PCD in accordance with the standard clinical diagnostic criteria at Peking Union Medical College Hospital (PUMCH). His sister is also affected by PCD, whereas both parents are healthy individuals. Blood samples from each individual of this family were collected. The proband of F2 is 39 years old male diagnosed with bronchiectasis at PUMCH. Samples of blood, bronchial tissue, and semen from this patient were collected. An informed consent of agreement to participate in this study was acquired from all individuals involved in this study. All methods in this study were approved by the Institutional Review Board committee at PUMCH.

2.2 Genetic analysis

gDNA was extracted from peripheral blood samples from the patient and his family by using a QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) via the standard methods described in the protocol. WES was performed on the Illumina PE150 platform with a sequence depth of 100×. The sequenced reads were aligned to the Genome Reference Consortium Human Build 37 (GRCh37/hg19) human assembly with the use of Burrows–Wheeler Aligner [14]. SAMtools and Sambamba were used to mark duplicate reads, analyze the coverage and depth of sequencing, and recognize single-nucleotide polymorphisms (SNPs), insertions, and deletions (indels) [15]. ANNOVAR was chosen as the annotation tool for SNPs and indels, including 1000 Genomes Project, NHLBI GO Exome Sequencing Project Exome Variant Server (ESP6500SI-V2), and GnomAD, to evaluate the frequency of mutations [16–18]. Mutations were excluded if the minor allele frequency (MAF) from any of the abovementioned databases was greater than 0.01 [19]. Variants within exonic regions or splicing regions (including 10 bp upstream or downstream of the exon) were retained. SIFT, Polyphen, MutationTaster, and CADD were chosen to analyze mutation pathogenicity [20–23]. The OMIM, ClinVar, HGMD, KEGG, PID, and REACTOME_PATHWAY databases were searched for functional annotation [24–29]. Sanger sequencing was further conducted using gDNA from the patients and their family members to verify the mutations and their parental origins.

2.3 Generation and maintenance of CFAP54 in-frame variant knock-in mouse model

The C57BL/6J-Cfap54^{em1C(M2512TQAEFLA)}/Gpt mouse model was built by GemPharmatech Co., Ltd (Jiangsu, China). The variant c.7535_7536delinCGCAGGCTGAATTCTTGGCA (p. M2512TQAEFLA) in Cfap54 was induced by CRISPR/Cas9 technology and homologous recombination. In brief, guide RNA was designed by CRISPR Design with a sequence of 5′-CCAGGACCACAGCTTGCATT-3′, synthesized, and cloned into the pGK1.1 plasmid. The donor DNA plasmid was cloned into the pMD18T plasmid (the sequence of the donor DNA is provided in the supplementary files). The donor DNA plasmid, along with sgRNA and Cas9, was microinjected into the pronuclei of fertilized one-cell-stage eggs from female mice in a C57BL/6J background. Positive F0 mice were identified by PCR and Sanger sequencing with PCR primers F: 5′-GATAGCTGGCTAGTTGTGTTCCTAC-3′ and R: 5′-CGTTAAGTCATCCATCAGAATGAGG-3′, and the sequencing primer R: 5′-CGTTAAGTCATCCATCAGAATGAGG-3′. The F0 mice were then mated with wild-type C57BL/6J mice to obtain stable inheritable heterozygous F1 mice. Homozygous mutant mice (Cfap54^ki/ki) were acquired by mating heterozygous mice. All protocols were approved by the Institutional Animal Care and Use Committee of Chinese Academy of Medical Science and Peking Union Medical College.

2.4 Transmission electron microscopy (TEM) of testis and trachea

Mice older than 8 weeks were anesthetized using 10% ethyl carbamate, and the testes and tracheae were sampled. The testes were cut into pieces smaller than 1 mm × 1 mm × 1 mm with clean scissors and immersed in fixative solution (2% paraformaldehyde and 2.5% glutaraldehyde/PBS mixed solution) for 4 h. The rib cage was cut open with scissors and forceps to expose the trachea. Fixative solution was injected into the lumen of the trachea to preliminarily fix respiratory cilia. The trachea was removed, cut into small pieces with clean scissors, and immersed in fixative solution for 4 h. After the samples were post-fixated in osmium tetroxide for 1 h, dehydrated through a graded ethanol series, and embedded in Epon 812, they were first cut into 600 nm sections and stained with tolonium chloride to localize the target structure under light microscopy. The selected regional samples were then cut into 70 nm sections and stained with uranyl acetate and lead citrate. TEM was performed using JEM-1400 Plus (JEOL) with an accelerating voltage of 80 kV. The images were acquired using a VELETA CCD camera (OSIS). Two wild-type and two homozygous mutant mice were involved in the TEM study. For each mouse, three different pieces of tissue were sampled in the trachea and testes.

2.5 High-speed video analysis of beat frequency of nasal respiratory cilia

The tracheae from wild-type and Cfap54^ki/ki mice were isolated in biological safety cabinets, cleaned, and washed using Hank’s Balanced Salt Solution (ThermoFisher, USA). They were then digested overnight (12 h–14 h) at 4 °C by using protease from Streptomyces griseus (Sigma–Aldrich, USA). The isolated nasal respiratory cells were cultured with Dulbecco’s modified Eagle’s medium (Gibco, USA) for 24 h to recover ciliary movement and then observed under a microscope with a magnification factor of 1000 at room temperature (24 °C–25 °C). Video recording was performed at a speed of 36 frames per second (fps), and the picture size was 212 × 212 pixels. The video was then divided into smaller blocks of 4 × 4 pixels, and the grayscale value changes in each block over time were acquired and converted into a frequency-dominant signal by using a fast Frontier transform to calculate the beat frequency [30]. The amplitude of the frequency from each block should be greater than [1] three times the amplitude of the background and [2] 99.5% of the maximum amplitude signal to reduce the noisy signal, similar to the strategy in CiliaFA software [31]. A ciliary beat frequency (CBF) histogram was generated with the frequency from each block of each video ranging from 1 Hz to 18 Hz to [1] exclude nonmotile background blocks [2], maintain a clinically relevant CBF range, and [3] maintain frequencies under the theoretically measured upper threshold (half of the fps of videos). The dominant frequency, which is the highest-population frequency in the frequency histogram, was determined and considered to be the windowed region in a strategy such as those in Yi’s research, CiliarMove, and Sisson-Ammons Video Analysis [32–34]. Six Cfap54^ki/ki mice paired with six wild-type mice were sampled. For each sample or cell cluster, 10 videos were recorded, and the CBF was compared using Mann–Whitney U test with a statistical significance (P value) of 0.05.

2.6 Statistical analysis

The difference in RNA expression was analyzed using two-tailed Student’s t-test in the minigene assay, mouse testes, and sperm samples from the patient and control. For RNA analysis in the bronchial mucosa of the patient and control, one-way ANOVA was adopted. The difference in the survival rate of mice was analyzed using log-rank test. For high-speed video analysis, the difference between the mutant and wild-type CBF was determined using Mann–Whitney U test. P values less than 0.05 were considered significant.

3 Results

3.1 Clinical features and genetic mutations in two independent patients with PCD

The proband from F1 is 32 years old male with a recurrent productive cough for 1 year, who was diagnosed with bronchiectasis through high-resolution computed tomography of the chest at PUMCH in 2014. His sister was also diagnosed with bronchiectasis at PUMCH in 2014, manifesting newborn pneumonia and suffering from rhinitis with sinusitis at her primary age. The patient was identified and verified to carry compound heterozygous mutations in CFAP54, a maternal frameshift deletion CFAP54: NM_001306084: c.2649_2657delinC (p. E883Dfs*47) in exon 20 and a paternal in-frame insertion c.7312_7313insCGCAGGCTGAATTCTTGG (p. T2438delinsTQAEFLA) in exon 53 through WES and Sanger sequencing (Fig.1–1C). Both mutations are novel in the GnomAD database. ACMG was chosen to classify the pathogenicity of the variants, and it showed the frameshift deletion to be pathogenic and the in-frame insertion to be likely pathogenic. The frameshift mutation was implicated to cause loss-of-function of CFAP54. The patient’s sister carrying the same variants also developed PCD symptoms and showed PCD-relevant family medical history. H&E staining and TEM of this proband’s sperm flagella were performed to investigate the morphology and the ultrastructure of the spermatozoa flagella. The proband’s sperm flagella were either short or absent, and the sperm head was congregated (Fig.1). The axonemal “9+2” ultrastructure of the patient’s sperm flagella was incomplete (Fig.1). In addition, the CFAP54 mRNA expression in the proband’s sperm significantly decreased compared with that in the normal control (Fig.1). The nasal nitric oxide (nNO) was 80 nL/min in the proband of F1, and this value is very close to the cutoff value of 77 nL/min shown in the PCD guideline [35]. The following CBF and qPCR analysis found no obvious difference between the normal and the proband of F1 (Fig. S1).

The proband of F2 is 39 years old male who had pneumonia starting from the age of 4 years, followed by recurrent productive bronchitis with cough. He had been diagnosed with asthma for 25 years and was found to have bronchiectasis through high-resolution computed tomography in 2020. He had moderate unreversible obstructive ventilatory dysfunction with 50% force expiratory volume in one second compared with the predicted value. The value of nNO was 114 nL/min in this proband, and the patient had two children. Through WES and Sanger analysis, two compound missense variants, c.4112A>C (p. E1371A) and c.6559C>T (p. P2187S), in CFAP54 (NM_001306084) were identified and assessed to be likely pathogenic by ACMG. TA cloning was performed, and two variants were confirmed to be located on different alleles (Fig. S2). On the basis of the WES results and clinical examinations (whole-blood cell analysis, hypersensitive C-reactive protein of liver and kidney, total immunoglobulins) of the patients, cystic fibrosis, immunodeficiency, and other causes of bronchiectasis were excluded.

3.2 In-vitro minigene assay to verify the pathogenicity of the frameshift mutation

The minigene assay was used to investigate the effect of the frameshift mutation, c.2649_2657delinC (p. E883Dfs*47) in CFAP54. Wild-type and mutant minigene plasmids were generated, containing the normal exon 20 sequence and c.2649_2657delinC mutant exon 20 sequence of CFAP54, respectively, between exon A and exon B in the pCAS2 plasmid (Fig.2). Compared with the wild-type plasmid, the mutation caused a reduction in the RNA expression of exon A + exon 20 + exon B transcripts (Fig.2), implicating the frameshift mutation as possibly pathogenic. The sequence of exon A + exon 20 + exon B transcripts was confirmed by Sanger sequencing (Fig.2).

3.3 Hydrocephalus in lateral ventricles of Cfap54^ki/ki mice

A knock-in mouse model was used to analyze the pathogenicity of the in-frame mutation, c.7312_7313insCGCAGGCTGAATTCTTGG (p. T2438delinsTQAEFLA) in CFAP54. It carries mutation c.7535_7536delinCGCAGGCTGAATTCTTGGCA (p. M2512TQAEFLA) in Cfap54, whose amino acid changes were identical to the changes in the patient (Fig.3). The knock-in mutation was verified by Sanger sequencing and qPCR, which showed that the insertion mutation caused a reduction in Cfap54 mRNA expression in the testes and brain of the mutant mice compared with the wild-type mice. Body observation and coronal MRI of the Cfap54^ki/ki mouse model showed a hydrocephalic phenotype with an enlarged cranial vault, accumulation of cerebrospinal fluid in lateral ventricles, and damaged brain. Half of the homozygous mice died spontaneously before sexual maturity (day 60), possibly because of severe hydrocephalus (Fig.3–3G). This finding indicated that the severity of hydrocephalus in Cfap54^ki/ki homozygotes was variable. Patients with PCD rarely develop hydrocephalus, but this symptom occurs relatively commonly in PCD mouse models in the C57BL/6J background [2,36].

3.4 Mucus accumulation in the sinus cavity and reduction in CBF of Cfap54^ki/ki mice

The mice were bred in a specific pathogen-free (SPF) environment; however, to simulate the living environment of patients with PCD, the mice were bred in a non-SPF environment and exposed to potential pathogens and bacteria for 2 weeks before assessing the sinusitis (Fig.4). Through MRI, the Cfap54^ki/ki mice showed mucus accumulation in the nasal passage, maxillary sinuses, or ethmoidal sinuses compared with the wild-type mice (Fig.4–4E). Nasal coronal sectioning and AB-PAS staining were performed to further confirm the phenotype. Five Cfap54^wt/wt, four Cfap54^ki/wt, and seven Cfap54^ki/ki mice were observed to diminish the phenotypic variability, and PCD-relevant phenotypes of mucus accumulation and distorted nasal septa appeared more frequently in Cfap54^ki/ki mice (Fig.4). Compared with wild-type CBF with a median value of 8.2 Hz (mean value of 8.2 Hz with a sample standard deviation of 3.5 Hz) at room temperature, the respiratory cilia from mutant mice showed a decrease in CBF, with a median value of 6.2 Hz (mean value of 7.0 Hz with a sample standard deviation of 3.1 Hz, Fig.4). The difference was significant, with a P value less than 1.00e–04 based on Mann–Whitney U test, further demonstrating that CFAP54 may be associated with the function of airway cilia. As for the beat pattern of the cilia, no difference was observed between wild-type and mutant mice (Supp_Movies).

3.5 Defects in sperm flagella of Cfap54^ki/ki mice

Seminiferous tubules of Cfap54^wt/wt and Cfap54^ki/ki mice, which survived beyond 8 weeks of age, were investigated by sectioning and H&E staining. The spermatids of wild-type mice elongated during spermiogenesis, and sperm flagella extended into the lumen of seminiferous tubules. By contrast, a reduction in the number of flagella was observed in the lumen of Cfap54^ki/ki homozygous seminiferous tubules, indicating that the elongation phase of spermatids was affected, although hooked sperm heads and nuclei could still occasionally be spotted in the epithelium (Fig.5–5D). The morphology of sperm from the epididymis was observed by H&E staining under microscopy, which further illustrated that sperm flagella were either absent or shortened in mutant mice (Fig.5). TEM was performed to investigate the ultrastructure of sperm flagella and respiratory motile cilia. In wild-type mice, the normal cross-sections of flagella were recognized as a “9+2” structure surrounded by outer dense fibers of sperm. By contrast, the sperm flagella from the testes of Cfap54^ki/ki mice were abnormally assembled and completely disorganized (Fig.5). However, no significant difference was observed in the ultrastructure of the respiratory motile cilia of Cfap54^wt/wt and Cfap54^ki/ki mice (Fig. S3 in supplemental files). The expression of Cfap54 mRNA in Cfap54^wt/wt and Cfap54^ki/ki mice was analyzed, and the results showed that this gene was highly expressed in the testes, lung, and brain of Cfap54^wt/wt mice, whereas the expression in the testes and brain from Cfap54^ki/ki mice exhibited a decreasing trend (Fig.5). However, no statistically significant difference in mRNA expression was found between the lung of Cfap54^wt/wt and Cfap54^ki/ki mice (Fig. S4). Dysfunction of CFAP54 may affect spermiogenesis and lead to infertility.

3.6 Two missense variants of CFAP54 identified in another patient with PCD

In another PCD-affected individual from F2, two compound missense variants, c.4112A>C (p. E1371A) and c.6559C>T (p. P2187S), in CFAP54 were identified and verified through WES and Sanger sequencing. Both missense CFAP54 variants were absent in the 1000 Genomes Project, gnomAD (East Asians), and Exome Aggregation Consortium. The amino acids encoded by these two variants are highly conserved among different species of vertebrate mammals, as indicated by Multalin. These two variants were predicted to be deleterious via SIFT, PolyPhen-2, and CADD except for the M2 variant that was predicted to be tolerated by SIFT (Tab.1). STRING analysis predicted that CFAP54 may be highly associated with CCDC39, which is essential for the assembly of inner dynein arms and the dynein regulatory complex in ciliary axonemes (Fig.6).

In addition, the CFAP54 mRNA expression in the bronchial tissue of the proband from F2 was significantly reduced or almost absent compared with that in three unrelated normal controls, and the expression of CFAP54 mRNA was reduced by ~40% in the sperm compared with that in normal male (Fig.7).

4 Discussion

This study identified two patients with PCD carrying compound heterozygous mutations in a possibly new pathogenic gene, CFAP54. In the proband from F1, the frameshift deletion c.2649_2657delinC (p. E883Dfs*47) was predicted to be disease causing by MutationTaster. As the open reading frame of CFAP54 contains 9486 nt, which increases the difficulties of either the generation of the CFAP54-expression plasmid or the utility of Western blotting, the minigene assay and qPCR were chosen to further investigate the pathogenicity of the mutation in vitro. In the minigene assay, the frameshift mutation caused the transcription of exon A + exon 20 + exon B to decrease significantly. Then, the pathogenicity of the in-frame mutation in the patient, c.7312_7313insCGCAGGCTGAATTCTTGG (p. T2438delinsTQAEFLA), was verified through a knock-in mouse model whose phenotypes of hydrocephalus, infertility, and mucus accumulation in nasal sinus cavities were very similar to those in PCD individuals. In the patient of F2, compound heterozygous variants of c.4112A>C (p. E1371A) and c.6559C>T (p. P2187S) in CFAP54 were also identified and predicted to be deleterious by SIFT, PolyPhen-2, and CADD. A notable detail that the patient of F2 had affected lung function and mildly affected sperm activity is in accordance with the CFAP54 mRNA expression levels in his lung and sperm, as shown in Fig.7. The missense variants in F2 may have less harmful effect on male reproductive system than frameshift and nonsense mutations. Furthermore, not all male individuals with PCD are infertile.

Usually, a decrease in nNO production is observed in patients with PCD, and it supports their diagnosis. However, on rare occasions, patients have nNO levels in the normal range [37]. Michael et al. also identified 12 out of 18 patients with PCD with RSPH1 mutations to have nNO levels above the diagnostic cutoff of 77 nL/min, with relatively milder clinical manifestations and intact ciliary ultrastructures [38,39]. In the present study, the nNO values were 80 nL/min in the proband of F1 and 114 nL/min in the proband of F2, both of whom showed mild clinical phenotypes in the respiratory system. In addition, the CBF of nasal respiratory cilia from the proband of F1 was normal. The results here, together with the results from other studies [38,39], may suggest a correlation between the nNO value and the severity of clinical symptoms of PCD.

No laterality defect or situs inversus phenotype was observed in mutant mice. This result can be elucidated by the CFAP54 associated with the C1d projection of the central apparatus in the “9+2” microtubule configuration. The general classification of motile cilia includes the motor cilium (“9+2”) and nodal cilium (“9+0”) on the basis of ultrastructural microtubular arrangement. The motile cilia have a central microtubular pair apparatus, transferring force during cilial beating and ensuring that cilial motility is maintained in the same direction along the airway. However, the nodal cilia lack the central pair of microtubules, and their motion is rotatory, which produces a leftward flow of extraembryonic fluid power in activating and establishing left-right sidedness and body laterality [7].

Hydrocephalus occurs relatively commonly in PCD mouse models in the C57BL/6J background [2,36]. For example, C57BL/6 mice developed hydrocephalus with dysfunctional Dnah5 (the homolog of DNAH5), Ccdc40, and Rsph9, but this symptom is rarely seen in patients with PCD [40–42]. The Cfap54 knockout mice in the C57BL/6J background died spontaneously before sexual maturity (8 weeks of age) and showed a sign of early mortality because of severe hydrocephalus [13]. However, the severity of hydrocephalus in the Cfap54 knock-in mouse model was variable, and only half of homozygotes died before 8 weeks of age. This result could be explained by the fact that in-frame mutation is a partial loss-of-function mutation and produces an incompletely functional product.

According to the results of high-speed video analysis, a slight reduction in CBF in the Cfap54^ki/ki mouse model was observed from 8.2 Hz to 6.2 Hz (~24.4% decrease). The trend is consistent with that in Cfap54^ko/ko mice, which showed a statistically significant decrease of ~14.2% from approximately 12.5 Hz to approximately 10 Hz [13]. Therefore, CFAP54 is likely to affect the function of the ciliary beat and be a disease-causing gene of PCD. The difference in CBF between the two mouse models could be caused by distinguished temperature and methods for measurement, considering that CBF increases with temperature and that the CBF at 37 °C for Cfap54^ko/ko mice could be higher than that at room temperature. At present, no standardized measurement and analysis procedure are available to calculate CBF, and normal CBF outcomes under various methods range from as low as 3.45 Hz to as high as 18.77 Hz [43–45]. The limits of upper and lower thresholds of CBF are often set empirically or by advice from medical researchers. In the present study, CBF was measured by dividing videos into smaller blocks and calculating the frequency of each block, which is a strategy of whole-field analysis and automatic analysis of the entire captured image without manual selection of regions of interest. As the human eyes tend to be attracted to fast-beating areas, artifacts introduced by manual selection bias can be avoided [43].

The Cfap54^ki/ki mouse model displayed a distinct ultrastructural deformity in axonemal arrangement in sperm flagella, which was similarly observed in the Cfap54 knockout mouse model and implied that the process of elongation of spermatids may be closely associated with the function of CFAP54. However, the absence of C1d projection in the respiratory cilia of the Cfap54 knockout mouse model was not observed in Cfap54^ki/ki mice. This result could be explained by the resolution of TEM or the methods of sampling. Applying techniques with higher resolution may be necessary, such as cryo-EM, which has been performed for the analysis of ciliary structural abnormalities [46,47]. The absence of C1d projection in the sperm of the patient in F2 was not observed (data not shown). Aside from the limitation of the TEM available in the study facility, the CFAP54 missense variants of c.4112A>C and c.6559C>T may have fewer effects on CFAP54 in sperm compared with nonsense and frameshift variants.

The Cfap54^ki/ki mouse model showed no significant abnormality in respiratory ciliary ultrastructure and a subtle reduction in CBF, which are similar to those in patients carrying recessive loss-of-function mutations in genes encoding the nexin dynein regulatory complex component of motile cilia, such as GAS8, CCDC164, and CCDC65 [48–50]. Ambiguous changes in CBF and ultrastructure are not rare. Approximately one-third of individuals with PCD have normal ciliary TEM cross-sections, and subtle deficiencies in ciliary beats are not readily identified by high-speed video microscopic evaluation, suggesting the difficulties in diagnosing patients with PCD [51,52].

Cfap54 and FAP54, the C. reinhardtii homolog, are previously known as components of C1d projection of the central apparatus, whose malfunction could decrease the beat frequency of motile cilia [13]. In the C. reinhardtii model, FAP54, a spring layer protein of C1d complex with four tetratricopeptide repeat domains, could directly bind to calcium-dependent calmodulin or indirectly be associated with calcium-dependent calmodulin through its interaction with FAP46 and FAP74 [53,54]. Other C1d component proteins, such as CFAP74 and CFAP221, have also been reported to be associated with PCD [8]. Recent studies showed that FAP221 and FAP74 with ASH domains interacting with the PF16 protein and the surface of the C1 microtubule, form a coordinate system on the tubulin lattice, stabilizing and elasticizing the central microtubule [53,54]. FAP221 is a rachis protein in the C1d complex; clusters FAP46, FAP54, FAP297, and CAM1, a calcium-dependent calmodulin, and the clustered protein will coordinate the activity of dynein isoforms and play a critical role in controlling CBF and waveform [12,53,55]. In 2008, Lee reported a mouse model with Cfap221/Pcdp1 loss of function showing PCD phenotypes [56]. Moreover, patients with PCD carrying compound heterozygous mutations in CFAP221 showed a change in the ciliary beating waveform from a simple backward and forward motion to an aberrant circular pattern and slight reduction in the beat frequency of motile cilia [57]. For the FAP74 protein in C. reinhardtii, the C1d projection of the central apparatus was missing when the expression of FAP74 was at a low level. FAP74 artificial microRNA mutants also have severely impaired motility [12]. Individuals with mutations in CFAP74 are affected by PCD and asthenoteratozoospermia or abnormalities of the sperm flagella [58,59]. All these reports implied that CFAP members associated with the central apparatus are critical to ciliary function, and loss of function of CFAP54 could affect the structure of the C1d projection as a component and then lead to PCD. However, further biochemical and cell biological studies are required to fully understand the true function of CFAP54.

The difference in defects in the structures of respiratory cilia and sperm flagella in mice, as revealed by TEM, implied that Cfap54 may exert diverse influences on ciliary and flagellar formation and maintenance. Similar symptoms have been observed in TTC12, a PCD-associated gene recently reported to cause the absence of single-headed inner dynein arms in respiratory cilia, defects in outer dynein arms, and a distinct subset of single-headed inner dynein arms in sperm flagella [60]. The Spef2 loss-of-function mouse model with spermatogenesis defects and PCD phenotypes also showed distinct cross-sections between cilia and sperm flagella, which may be associated with the dysfunction of intraflagellar transport [61]. This finding can be explained by the differences in components of the ultrastructural protein complex between airway cilia and spermatozoa. Moreover, the flagella in C. reinhardtii may mimic the sperm flagellar phenotype but not the ciliary phenotype, thus demonstrating a limitation of the C. reinhardtii model.

In conclusion, CFAP54 was found to be a new PCD-associated gene identified in two individuals with PCD for the first time. The pathogenicity of the CFAP54 compound heterozygous mutations c.2649_2657delinC and c.7312_7313insCGCAGGCTGAATTCTTGG in F1 was investigated through an in-vitro minigene assay and an in-vivo nonframeshift mutation knock-in mouse model, respectively. The reduced mRNA expression in the lung and sperm of the patient harboring CFAP54 missense variants c.4112A>C and c.6559C>T in F2 was also validated. The findings help increase the landscape of the PCD-relevant gene spectrum and contribute to the identification of target genes for genetic PCD testing.

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