Two Novel Compound Heterozygous Mutations of CFAP65 in a Pediatric Patient With Primary Ciliary Dyskinesia

The patient was an 8-year-old Han Chinese boy who had suffered from recurrent pneumonia for 1 year and 8 months. The initial episode of pneumonia occurred at the age of 6 years, presenting with fever and cough, accompanied by sputum production and nasal congestion. The chest X-ray revealed patchy opacities in the right lower lung. He was diagnosed with pneumonia and admitted to the local hospital due to the failure of oral antibiotic therapy. He was treated with intravenous antibiotics and budesonide nebulization. After 12 days of treatment, his symptoms improved. After discharge, he still experienced recurrences of fever and cough with sputum, which were partially relieved with oral medications at home. He had been hospitalized with a diagnosis of pneumonia after his symptoms failed to respond to oral antibiotics, on two occasions (11 months and 2 months before the current admission). The patient presented persistent productive cough. One month ago, due to nasal congestion and rhinorrhea, a sinus CT scan showed sinusitis, and mometasone furoate nasal spray was administered. Beyond the chronic respiratory symptoms, review of his systems was unremarkable. There was no reported otitis media, hearing loss, or symptoms involving other systems. No decrease in physical activity tolerance was observed. The patient was delivered at term via cesarean section with a birth weight of 3.0 kg. He was diagnosed with aspiration pneumonia shortly after birth and was hospitalized in the neonatal unit for 15 days. Nasal congestion and rhinorrhea had been present since infancy (within the first 6 months of life). The patient had experienced frequent respiratory infections since infancy, with respiratory symptoms occurring only during the infections. It was not until the age of 6 that chronic respiratory symptoms emerged. His parents are non-consanguineous, and his elder sister was healthy. On physical examination, vital signs on presentation were as follows: temperature 36.20°C, heart rate 98 beats per minute, respiratory rate 24 breaths per minute, and blood pressure 90/60 mmHg. He was alert and afebrile, with appropriate nutritional status and normal development. Respiratory auscultation revealed coarse breath sounds with rales. There were no signs of cyanosis or clubbing. After admission, a series of diagnostic examinations were performed to evaluate the cause of chronic productive cough and recurrent pneumonia. HRCT of the chest showed patchy opacities and bronchiectasis in the right middle lobe (Figure 1A). Echocardiography and audiometry results were normal. Pulmonary function tests indicated mild obstructive ventilatory impairment, with spirometry revealing FVC of 87.5% predicted, FEV1 of 83.2% predicted, and FEV1/FVC ratio of 93.9%. Bronchoscopy revealed endobronchial inflammation with abundant secretions. Comprehensive microbiological studies were performed on both sputum and bronchoalveolar lavage fluid samples to investigate potential pathogens. These included cultures for bacterial, fungal, and Mycobacterium tuberculosis, molecular testing for Mycoplasma pneumonia, and serological testing for IgM antibodies against common respiratory viruses. No definitive evidence of infection was identified. Given the recurrent pneumonia, bronchiectasis, sinusitis, history of neonatal pneumonia, and persistent nasal symptoms, primary ciliary dyskinesia (PCD) was considered as the possible diagnosis. At the same time, a sweat chloride test was normal, ruling out cystic fibrosis. Normal results of the immunoglobulin assay and analysis of circulating B, T, and natural killer cells indicate that immunodeficiency is unlikely. To determine the diagnosis of PCD, the following investigations were performed: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM) of ciliary ultrastructure, high-speed video microscopy analysis (HSVA) of ciliary beat, and genetic testing. nNO was measured by chemiluminescence analyser using the velum closure technique. During the current hospitalization, the nNO was 9 nL/min, which was significantly lower than the diagnostic threshold of 77 nL/min for PCD. A repeat measurement was performed after 1 month to confirm a persistently low level. The value remained significantly reduced at 36 nl/min, with the patient at baseline respiratory status during both tests. HSVA of bronchial mucosa brushing showed ciliary beating dyskinesia with circular pattern (Supporting Information S1: Videos 1, 2). One month later, a repeat HSVA performed on a nasal brushing specimen confirmed the same abnormal ciliary beating pattern. TEM of bronchial mucosa tissues revealed the defects in the radial spokes and mislocation of the central apparatus of microtubules (Figure 1B). All specimens mentioned above were collected while the patient was at baseline respiratory status. Whole exome sequencing was performed using the Illumina platform to investigate the genetic cause of PCD. No pathogenic or likely pathogenic variants were identified in known PCD-associated genes. Bi-allelic nonsynonymous variants (NM_194302; exon29: c.4568 T > C, p.L1523P; NM_194302; exon16: c.2765 G > A, p.R922Q) of CFAP65 were identified. Both variants were absent or extremely rare in population databases (p.L1523P: not present in any databases; p.R922Q: genomeAll AF = 0.0005, genomeEas AF = 0.0026). Co-segregation analysis via Sanger sequencing confirmed that the p.L1523P and p.R922Q variants were maternally and paternally inherited, respectively (Figure 2A). According to ACMG criteria, both two variants were scored as uncertain significance. To assess the effect of missense mutations on protein structure, wild-type and mutant protein models were constructed using SWISS-MODEL software. For the c.4568 T > C (p.L1523P) mutation, in the wild-type protein structure, there are two hydrogen bonds between L1523 and G1610, whereas in the MutL1523P variant, the P1523 side chain forms a benzene ring structure, and one hydrogen bond between P1523 and G1610 is broken, potentially leading to local structural instability of the mutant. For the c.2765 G > A (p.R922Q) mutation, in the wild-type protein structure, there are three hydrogen bonds between R922 and E919 and two hydrogen bonds between R922 and V917, while in the MutR922Q variant, two hydrogen bonds between Q922 and E919 and one hydrogen bond between Q922 and V917 are broken, which may cause local structural instability (Figure 2B). Detailed materials and methods for high-speed video microscopy, TEM analysis, whole exome and Sanger sequencing, and variant analyses are provided in the Supporting File. This study has been approved by the Ethical Committee of Beijing Children's Hospital (ethical approval number: [2024]-E-132-R). Participants included in the study provided informed consent. PCD is a rare, heterogeneous inherited disorder leading to chronic sinopulmonary, laterality defects, and subfertility. Now, approximately 54 genes were identified to cause PCD [1]. In the current case, the child demonstrated typical symptoms of PCD, with the significantly reduced nNO level, abnormal respiratory ciliary beat patterns, as well as the ultrastructural defects in cilia. A diagnosis of PCD was established, and two novel compound heterozygous mutations of CFAP65 were identified in this case. A series of genetic variants of CFAP65 has been reported, leading to multiple morphological abnormalities (MMAF) in humans. Wang et al. and Lu et al. reported infertile men exhibiting typical MMAF phenotypes, as well as PCD-like symptoms, but did not describe defects in respiratory cilia [2, 3]. The structure of sperm tails is nearly identical to that of respiratory cilia. Consequently, males with PCD often experience diminished fertility due to reduced sperm motility, with reports indicating that nearly 100% of adult males with PCD are infertile. However, this phenotype is typically not observed in childhood. In this case, the ultrastructural defects in respiratory cilia were similar to those previously reported in sperm tails [2, 3]. The circular ciliary beat pattern observed was similar to that seen in cases with defects in the RSH4A or RSPH9 genes [4], which encode radial spoke head proteins [5]. The protein encoded by CFAP65 is located on one of the central apparatus components [6, 7], which could explain the ultrastructural defects and abnormal beat pattern of cilia. Additionally, as expected, no laterality defects were observed in this case, as dysfunction of the central apparatus or radial spokes is not anticipated to disrupt nodal cilia function responsible for establishing left-right body asymmetry. This case expands the genetic spectrum of PCD and underscores the importance of considering genes traditionally linked to infertility in patients with convincing clinical signs of PCD, even in childhood. Further functional studies are warranted to fully elucidate the role of CFAP65 in respiratory cilia and validate its pathogenicity in PCD. In summary, this case suggests that CFAP65 gene mutations can cause PCD, and is the first to describe respiratory cilia defects, including both ultrastructural abnormalities and beat pattern defects, which corresponded with each other. Hao Wang: writing − original draft, data curation, methodology, visualization. Lina Wang: writing − original draft, formal analysis, methodology. Xiuyun Liu: data curation, writing − review, and editing. Baoping Xu: writing − review and editing, conception, supervision. Kunling Shen: writing − review and editing, conception, supervision. We would like to acknowledge the patient and his family members for participating in our research. National Nature Science Foundation of China (32000590). This study has been approved by the Ethical Committee of Beijing Children's Hospital (ethical approval number: [2024]-E-132-R). Participants included in the study provided informed consent. The authors declare no conflicts of interest. All data and materials are available from the corresponding author on reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.