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Introduction: Primary ciliary dyskinesia (PCD) is a rare heterogeneous disease caused by abnormalities in motile cilia. In this case report, we first analyzed the clinical and genetic data of a proband who was suspected of having PCD on the basis of her clinical and radiological findings. Methods: Whole-exome sequencing was performed, and a variant in the RSPH4A gene was identified in the proband. Sanger sequencing was used for validation of RSPH4A variants in the proband, her sister, her daughter and her parents. Finally, the phenotypic features of the patient were analyzed, and the current literature was reviewed to better understand the gene variants in PCD related to hearing loss and the clinical manifestations of the RSPH4A variant in PCD. Results: The chief clinical symptoms of this proband included gradual mixed hearing loss, otitis media, anosmia, sinusitis, recurrent cough and infertility. Her DNA sequencing revealed a novel homozygous T to C transition at position 1321 within exon 3 of RSPH4A according to genetic testing results. This variant had never been reported before. The homozygous variant resulted in an amino acid substitution of tryptophan by arginine at position 441 (p.Trp441Arg). The same variant was also found in the proband\'s sister, and a heterozygous pathogenic variant was identified among immediate family members, including the proband\'s daughter and parents. Discussion: A literature review showed that 16 pathogenic variants in RSPH4A have been reported. Hearing loss had only been observed in patients with the RSPH4A (c.921+3_6delAAGT) splice site mutation, and the specific type of hearing loss was not described.

The radial spoke head protein 4 homolog A (RSPH4A) gene is one of more than 50 genes that cause Primary ciliary dyskinesia (PCD), a rare genetic ciliopathy. Genetic mutations in the RSPH4A gene alter an important protein structure involved in ciliary pathogenesis. Radial spoke proteins, such as RSPH4A, have been conserved across multiple species. In humans, ciliary function deficiency caused by RSPH4A pathogenic variants results in a clinical phenotype characterized by recurrent oto-sino-pulmonary infections. More than 30 pathogenic RSPH4A genetic variants have been associated with PCD. In Puerto Rican Hispanics, a founder mutation (RSPH4A (c.921+3_921+6delAAGT (intronic)) has been described. The spectrum of the RSPH4A PCD phenotype does not include laterality defects, which results in a challenging diagnosis. PCD diagnostic tools can combine transmission electron microscopy (TEM), nasal nitric oxide (nNO), High-Speed Video microscopy Analysis (HSVA), and immunofluorescence. The purpose of this review article is to provide a comprehensive overview of current knowledge about the RSPH4A gene in PCD, ranging from basic science to human clinical phenotype.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disease characterized by motile cilia dysfunction with a prevalence of 1 in 16,309 individuals in Hispanic populations. In Puerto Rico, the prevalence of PCD is unknown. Diagnosis of PCD in Puerto Rico is challenging due to the lack of diagnostic technology. Algorithms for PCD diagnosis include clinical history, genetic testing, ciliary biopsy, and nasal Nitric Oxide (nNO) levels. For the first time, this study successfully implemented and measured the nNO levels in subjects with the RSPH4A (c.921+3_921+6del (intronic)) as a diagnostic tool to complement the current algorithm for PCD diagnosis on the island. The nNO level differentiated homozygous subjects with PCD due to the RSPH4A (c.921+3_921+6del (intronic)) founder mutation compared to healthy gender-age matched controls and subjects with VUS or negative genetic testing for PCD. The acquisition of state-of-the-art diagnostic tools such as nNO positively impacted and expanded our current PCD diagnostic capabilities in Puerto Rico for our founder genetic mutation. The addition of nNO technology promotes earlier disease screening and recognition for patients with PCD on the island. The access to nNO helped us to properly characterize the PCD diagnosis for patients with the RSPH4A (c.921+3_921+6del (intronic)). As a result, our findings will allow us to be part of the national PCD foundation registry and represent Puerto Rican Hispanics in future PCD multicentric clinical trials.

Background: The radial spoke head component 4A (RSPH4A) is involved in the assembly of radial spokes, which is essential for motile cilia function. Asthenoteratozoospermia in primary ciliary dyskinesia (PCD) related to RSPH4A variants has not been reported. Materials and Methods: RSPH4A variants were identified and validated using whole-exome and Sanger sequencing in three unrelated Chinese families. High-speed video microscopy analysis (HSVA) was performed to measure the beating frequency and pattern of nasal cilia of the patients and healthy control. Papanicolaou staining and computer-aided sperm analysis were performed to analyze the morphology and motility of the sperm in patient 1. Immunofluorescence was adopted to confirm the structure deficiency of sperm and nasal cilia. Results: Patient 1 from family 1 is a 22-year-old unmarried male presented with bronchiectasis. Semen analysis and sperm Papanicolaou staining confirmed asthenoteratozoospermia. Novel compound heterozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.1774_1775del, p.(Leu592Aspfs*5) were detected in this patient. Patients 2 and 3 are from two unrelated consanguineous families; they are both females and exhibited bronchiectasis and infertility. Two homozygous RSPH4A variants c.2T>C, p.(Met1Thr) and c.351dupT, p.(Pro118Serfs*2) were detected, respectively. HSVA showed that most of the cilia in patients 1 and 3 were with abnormal rotational movement. The absence of RSPH4A and RSPH1 in patient 1\'s sperm and patient 3\'s respiratory cilia was indicated by immunofluorescence. Patient 2 died of pulmonary infection and respiratory failure at the age of 35 during follow-up. Conclusion: Dysfunctional sperm flagellum and motile cilia in the respiratory tract and the fallopian tube were found in patients with RSPH4A variants. Our study enriches the genetic spectrum and clinical phenotypes of RSPH4A variants in PCD, and c.2T>C, p.(Met1Thr) detected in our patients may be a hotspot RSPH4A variant in Chinese.

Primary ciliary dyskinesia (PCD) has been linked to more than 50 genes that cause a spectrum of clinical symptoms, including newborn respiratory distress, sinopulmonary infections, and laterality abnormalities. Although the RSPH4A (c.921+3_6delAAGT) pathogenic variant has been related to Hispanic groups with Puerto Rican ancestry, it is uncertain how frequently other PCD-implicated genes are present on the island. A retrospective chart review of n = 127 genetic reports from Puerto Rican subjects who underwent genetic screening for PCD variants was conducted from 2018 to 2022. Of 127 subjects, 29.1% subjects presented PCD pathogenic variants, and 13.4% were homozygous for the RSPH4A (c.921+3_6delAAGT) founder mutation. The most common pathogenic variants were in RSPH4A and ZMYND10 genes. A description of the frequency and geographic distribution of implicated PCD pathogenic variants in Puerto Rico is presented. Our findings reconfirm that the presence of PCD in Puerto Rico is predominantly due to a founder pathogenic variant in the RSPH4A (c.921+3_6delAAGT) splice site. Understanding the frequency of PCD genetic variants in Puerto Rico is essential to map a future genotype-phenotype PCD spectrum in Puerto Rican Hispanics with a heterogeneous ancestry.

Genetic mutations in >50 genes, including RSPH4A, can lead to primary ciliary dyskinesia (PCD). RSPH4A mutations affect radial spokes, which alter the configuration of the ciliary ultrastructure and lead to chronic oto-sinopulmonary disease. The RSPH4A [c.921+3_6delAAGT] founder mutation was described as one cause of PCD without laterality defects in Puerto Rico. The average Puerto Rican genetic composition includes 64% European, 21% African ancestral, and 15% Native-American or Taino, a native tribe in the Caribbean at the start of the European colonization, genes. Due to the relatively elevated Taino ancestry on the island, it might have contributed to the endemicity of the RSPH4A [c.921+3_6delAAGT] splice site mutation. However, the ancestry of this mutation is still not confirmed. This article describes the two pediatric PCD cases with the Puerto Rican foundermutationand reports an ancestral haplotype analysis of the RSPH4A [c.921+3_6delAAGT] splice site mutation. A median-joining haplotype network was constructed with the genome sequence data from 104 Puerto Rican subjects in the 1000 Genomes Project (1000GP). This study found that the RSPH4A [c.921+3_6delAAGT] splice site mutation was carried to Puerto Rico from Europe by conquistadors or shortly after the conquest and that it gained frequency on the island through genetic drift fueled by a subsequent population expansion.

Primary ciliary dyskinesia (PCD) is a rare, heterogeneous ciliopathy resulting in chronic oto-sino-pulmonary disease, bronchiectasis, newborn respiratory distress, and laterality defects. PCD diagnosis can be achieved by following diagnostic algorithms that include electron microscopy, genetics, and ancillary testing. Genetic mutations in more than 45 genes, including RSPH4A, can lead to PCD. RSPH4A mutations located on chromosome six, affect radial spokes and results in central complex apparatus abnormalities. The RSPH4A [c.921 + 3_6delAAGT] founder mutation was described as one cause of PCD without laterality defects in Puerto Rico. Additionally, there are further diagnostic challenges present in the Puerto Rican population to diagnose PCD. We describe the demographics, clinical features, and RSPH4A genetic variants in 13 patients with clinical PCD affecting 11 Puerto Ricans from unrelated families.

Radial spokes are structurally conserved, macromolecular complexes that are essential for the motility of 9 + 2 motile cilia. In Chlamydomonas species, mutations in radial spoke proteins result in ciliary motility defects. However, little is known about the function of radial spoke proteins during embryonic development. Here, we investigated the role of a novel radial spoke protein, leucine-rich repeat containing protein 23 (Lrrc23), during zebrafish embryonic development. Mutations in lrrc23 resulted in a selective otolith formation defect during early ear development. Similar otolith defects were also present in the radial spoke head 3 homolog ( rsph3) and radial spoke head 4 homolog A ( rsph4a) radial spoke mutants. Notably, the radial spoke protein mutations specifically affected ciliary motility in the otic vesicle (OV), whereas motile cilia in other organs functioned normally. Via high-speed video microscopy, we found that motile cilia formation was stochastic and transient in the OV. Importantly, all the motile cilia in the OV beat circularly, in contrast to the planar beating pattern of typical 9 + 2 motile cilia. We identified the key time frame for motile cilia formation during OV development. Finally, we showed that the functions of radial spoke proteins were conserved between zebrafish and Tetrahymena. Together, our results suggest that radial spoke proteins are essential for ciliary motility in the OV and that radial spoke-regulated OV motile cilia represent a unique type of cilia during early zebrafish embryonic development.-Han, X., Xie, H., Wang, Y., Zhao, C. Radial spoke proteins regulate otolith formation during early zebrafish development.

Occupational exposures to the diketone flavoring agent, diacetyl, have been associated with bronchiolitis obliterans, a rare condition of airway fibrosis. Model studies in rodents have suggested that the airway epithelium is a major site of diacetyl toxicity, but the effects of diacetyl exposure upon the human airway epithelium are poorly characterized. Here we performed quantitative LC-MS/MS-based proteomics to study the effects of repeated diacetyl vapor exposures on 3D organotypic cultures of human primary tracheobronchial epithelial cells. Using a label-free approach, we quantified approximately 3400 proteins and 5700 phosphopeptides in cell lysates across four independent donors. Altered expression of proteins and phosphopeptides were suggestive of loss of cilia and increased squamous differentiation in diacetyl-exposed cells. These phenomena were confirmed by immunofluorescence staining of culture cross sections. Hyperphosphorylation and cross-linking of basal cell keratins were also observed in diacetyl-treated cells, and we used parallel reaction monitoring to confidently localize and quantify previously uncharacterized sites of phosphorylation in keratin 6. Collectively, these data identify numerous molecular changes in the epithelium that may be important to the pathogenesis of flavoring-induced bronchiolitis obliterans. More generally, this study highlights the utility of quantitative proteomics for the study of in vitro models of airway injury and disease.