Two Japanese Pediatric Patients With Primary Ciliary Dyskinesia Caused by Loss-of-Function Variants in the CCNO gene

Primary ciliary dyskinesia (PCD) is a rare congenital disorder caused by pathogenic variants of genes related to cilia. Here, we report two Japanese pediatric patients with PCD caused by pathogenic compound heterozygous variants in the cyclin O (CCNO) gene (Case 1, NM_021147.4:c.[262C>T];[781delC], p.[Gln88Ter];[Leu261fs]; Case 2, c.[262C>T];[c.248_252dupTGCCC], p.[Gln88Ter];[Gly85fs]). The clinical symptoms of the patients were varied. Neither of the patients had situs inversus. Transmission electron microscopy of the respiratory cilia from the nasal mucosa in Case 1 showed a remarkable reduction of cilia and the few residual cilia had central pair defects and microtubular disorganization.


Introduction
Primary ciliary dyskinesia (PCD) is a congenital disorder caused by pathogenic variants of genes related to cilia.Most cases are inherited in an autosomal recessive manner, and more than 50 PCD causative genes have been reported [1].Disease severity varies with the causative gene and variant [1,2].PCD is characterized by persistent productive cough due to inefficient respiratory mucociliary clearance caused by ciliary beating disorders [1].The varied symptoms of PCD include recurrent chronic airway infections, otitis media with effusion, infertility, and situs inversus [1].
Dynein axonemal heavy chain 5 (DNAH5), dynein axonemal intermediate chain 1 (DNAI1), and DNAH11 are the most common causative PCD genes among most ethnicities, accounting for 15%-29%, 2%-10%, and 6%-9% of PCD patients, respectively, in European and American populations [1,2].We reported recently that dynein regulatory complex subunit 1 (DRC1) and DNAH5 are the most frequent causative genes in Japanese PCD patients [3][4][5].Cases with variants in cyclin O (CCNO) are rare and account for less than 2% of PCD patients [1].Here, we report for the first time two Japanese pediatric cases of PCD in whom CCNO was considered to be the causative gene by genetic testing.

Case Presentation Case 1
A 5-year-old boy was referred to our department for a persistent productive cough.He was born at 39 weeks.He had rhinorrhea and a productive cough immediately after birth and was admitted to the neonatal intensive care unit due to neonatal respiratory distress syndrome.He had persistent rhinosinusitis and refractory otitis media with effusion.He did not have situs inversus or a congenital heart defect.He did not have hydrocephalus.His parents and 9-year-old brother did not present with any symptoms of PCD.His PICADAR (PrImary CiliARy DyskinesiA Rule) score was 8 based on seven diagnostic questions to predict the likelihood of having PCD, indicating a 66% possibility of PCD.Rhonchi breath sounds were heard in both lungs with mild intercostal retraction.His heart rate was 120 bpm, and SpO 2 was 98% on room air.
Haemophilus influenzae (2+) was identified by a bronchoscopic culture.A nasal nitride oxide (nNO) test was not performed.He had residual fluid in the bilateral middle ears (Figure 1A, 1B).Mucus secretions were observed in the bilateral nasal cavities on nasal endoscopy (Figure 1C, 1D).Chest X-rays immediately after birth and at 10 months had shown a shadow in the right upper lobe (Figure 1E, 1F).Chest computed tomography showed consolidation in the right upper lobe (Figure 1G-1I).Transmission electron microscopy of respiratory cilia from the nasal mucosa showed either a complete absence or severely decreased numbers of cilia (Figure 2).Exome sequencing performed in 32 known PCD genes [4] identified two heterozygous variants in CCNO (NM_021147.4:c.[262C>T];[781delC],p.[Gln88Ter];[Leu261fs]); causative pathogenic variants in other PCD genes could not be found.Both variants were predicted to be loss-of-function mutations, and neither has been reported previously.Sanger sequencing of Case 1 and his parents confirmed the presence of compound heterozygous variants (Figure 3).His mother carried the former variant and his father carried the latter variant.

Case 2
The patient was a 13-year-old boy with a persistent wet cough whose blood samples were sent to our department for genetic testing.He was born at full term and was admitted to a neonatal intensive care unit due to pneumonia on the second day after birth.He had been suffering from refractory pneumonia (Figure 4A), atelectasis (Figure 4B) and chronic rhinosinusitis (Figure 4C).He was hospitalized due to bacterial pneumonia at 13 years of age, and bronchiectasis was noted at that time (Figure 4D).He did not have situs inversus or a congenital heart defect.He did not have hydrocephalus.His parents and 12-year-old sister did not present with the same symptoms.His PICADAR score was 7, indicating a 44% possibility of PCD.Breath sounds were diminished in the left lung and coarse crackles were heard in the right lower lung.His body temperature was 38.5°C, SpO 2 88% (room air), heart rate was 120 bpm, and respiratory rate was 26 breaths/min.Forced vital capacity (FVC) was 3.57 L (94.2%), Forced expiratory volume in the first second (FEV 1 ) was 2.64 L (77.0%), FEV 1 % was 73.95% at the time of one-year follow-up.Streptococcus pneumoniae (PSSP) (1+), Neisseria species (small amount), and Haemophilus parainfluenzae (small amount) were identified by sputum culture.A nasal nitride oxide (nNO) test was not conducted, and exhaled nitric oxide was 8ppb.Exome sequencing performed in 32 known PCD genes [4] identified two heterozygous variants in CCNO (NM_021147.4:c.[262C>T];[c.248_252dupTGCCC],p.[Gln88Ter];[Gly85fs]); causative pathogenic variants in other PCD genes could not be found.Sanger sequencing confirmed that the mother carried the former variant, while the father carried the latter one (Figure 5).Findings from both cases are summarised in Table 1

Discussion
To our knowledge, 55 PCD cases caused by CCNO worldwide have been published in the literature (Supplementary Material 1).This is the first report of PCD in Japanese patients caused by pathogenic variants in CCNO.Wallmeier et al. first reported disease-causing CCNO variants in 16 patients with chronic destructive lung disease due to insufficient airway clearance and identified a homozygous variant (c.248_252dupTGCCC, p.Gly85Cysfs*10) in a large consanguineous Kuwaiti family [6], which is the same variant found in Case 2 in the present report.The same variant was also reported recently in a Chinese female PCD patient [7].In addition, we identified two novel variants, the nonsense mutation c.262C>T (rs1272978797) in Cases 1 and 2, and the frameshift deletion mutation c.781delC in Case 1 (Supplementary Material 2).Neither of these variants has been reported in PCD patients.
CCNO is a cyclin-like protein and has been predicted to function in the cell cycle and transcriptional control.It plays a critical regulatory role in deuterosome-mediated centriole amplification during multiciliogenesis [8].Multiciliated cells have specific procentriole nucleation centers called deuterosomes, where the massive amplification of centrioles is achieved [9].During cilia biogenesis, centrioles amplify and dock to the apical cell membrane form basal bodies and serve as a platform and rootlet for the outgrowth of ciliary axonemes [10].Ccno-deficient multiciliated cells fail to generate sufficient numbers of functional deuterosomes, resulting in a marked reduction and maturation defects of centrioles.The centrioles fail to localize correctly during their transition to basal bodies.As a result, CCNO-deficient cells show reduced numbers of motile cilia on their apical surface, leading to mucociliary clearance defects [8,11].
Consistent with a previous report [6], transmission electron microscopy in Case 1 in the present report showed that the basal bodies and rootlets were mislocalized within the cytoplasm of nasal ciliated cells, not at the apical cell region, suggesting a defect in centriole generation and basal body migration.Compared with approximately 200 cilia per ciliated cell in healthy controls, cases with CCNO defects have only one or two cilia per cell [6,12].This CCNO deficiency may lead to ultrastructural defects of axonemes [8].Case 1 had central pair defects and microtubular disorganization.These ciliary ultrastructural defects might be caused primarily by CCNO deficiency or as a result of secondary defects due to increased susceptibility to inflammation or infection.
Our two pediatric cases presented with severe respiratory symptoms early in life.CCNO cases reportedly show rapid deterioration in lung function, and CCNO pediatric cases are more likely to have bronchiectasis, with an incidence of 89% [13].In contrast, the incidence of bronchiectasis among other pediatric PCD cohorts has been reported as 50%-75% [14] and 38% [15].CCNO cases also have a higher incidence of neonatal respiratory distress syndrome [13][14][15].
CCNO deficiency causes defective ciliary function of ependymal multi-ciliated cells, reducing the intraventricular transport of cerebrospinal fluid and leading to hydrocephalus.The clinical symptoms in humans and mice appear to vary; hydrocephalus is not common in humans and mice do not show obvious respiratory problems [8].Ccno deletion causes 57% of the mutant mice to develop hydrocephalus [8]; a systematic analysis of CCNO cases revealed that hydrocephalus occurs in 10% of human patients [13].Our two cases did not have hydrocephalus.They also did not have situs inversus, probably because the axonemal motor proteins of the few residual cilia are expressed correctly leading to intact ciliary beating during organ asymmetry at the node in early-somite stage embryos [6].

Conclusions
We encountered two Japanese pediatric patients with PCD caused by pathogenic variants in CCNO.The clinical symptoms of the patients were varied, and neither of the patients had situs inversus.Case 1 in the present report showed a remarkable reduction of cilia and an abnormal ciliary ultrastructure of mislocalized basal bodies and rootlets by transmission electron microscopy, suggesting a defect in centriole generation and basal body migration.We also observed and first reported ciliary ultrastructural defects of axonemes in the CCNO case.In addition, we identified two novel variants, namely CCNO NM_021147.4:c.262C>T(p.Gln88Ter) and c.781delC (p.Leu261fs).

Appendices
Supplementary material 1  All the variants are marked with arrows.All the variants are classical loss-of-function mutations.Disease-causing variants reported in the literature (in italics) [6] are also shown in the figure.

FIGURE 1 :
FIGURE 1: Imaging findings in Case 1. (A, B) Residual fluid and air-fluid levels in the bilateral middle ears.(C, D) Mucus secretion in the bilateral nasal cavities.(E) Chest X-rays immediately after birth and (F) at 10 months showed a shadow (arrows) in the right upper lobe.There was no situs inversus.(G) Chest computed tomography of the upper, (H) middle, and (I) lower lobes showed consolidation (arrow) in the right upper lobe.

FIGURE 2 :
FIGURE 2: Transmission electron microscopy findings of a biopsy specimen from the nasal mucosa.
(A) Longitudinal and (B) cross sections of Case 1 showing either complete absence or a remarkable reduction of cilia; (A) the residual cilium has microtubular disorganization; (B) the residual cilium lacks central pairs.(C, D) Cilia from healthy individuals (15-year-old female and 14-year-old male non-PCD controls) show the normal number of cilia; the cilia consist of one central pair of microtubules surrounded by nine pairs of well-arranged peripheral microtubules.(E) Nasal ciliated cells from Case 1.The basal bodies (arrows) and attached rootlets (circles) are mislocalized in the cytoplasm, far from the apical cell region.(F) Nasal ciliated cells from a healthy individual.The basal bodies attach to the rootlets and dock to the apical cell regions.

FIGURE 3 :
FIGURE 3: Family pedigree of Case 1 with the results of Sanger sequencing.Compound heterozygous variants in CCNO are shown.

FIGURE 4 :
FIGURE 4: Imaging findings in Case 2. (A) Chest X-ray at 5 years of age showed infiltrative shadows (circles) in the bilateral lower lung.(B) Chest X-ray showed atelectasis in the left lower lobe (circle).(C) Nasal X-ray at 13 years of age showed thickened mucosa of the bilateral maxillary sinuses (arrows).(D) Chest computed tomography at 13 years of age showed bronchiectasis (circle).

FIGURE 5 :
FIGURE 5: Family pedigree of Case with the results of Sanger sequencing.Compound heterozygous variants in CCNO are shown.

FIGURE 6 :
FIGURE 6: Schematic of CCNO gene and predicted consequences of the variants at the protein level.

TABLE 1 : Clinical characteristics and examination findings of patients.
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FCV: Forced vital capacity; FEV 1 :Forced expiratory volume in the first second