Maimoona A. Zariwala

Introduction: Primary ciliary dyskinesia (PCD), a disorder of motile ciliary dysfunction causing chronic respiratory infections, may also rarely present with aspects of non‐motile ciliary dysfunction, including retinitis, central nervous system malformations, skeletal dysplasia, and sensorineural hearing loss. However, congenital microcephaly has not been previously associated with PCD. Methods: We identified three children with congenital microcephaly and chronic respiratory disease consistent with PCD. Comprehensive PCD diagnostic testing and whole exome sequencing were performed. Characterization of nasal epithelial cells after expansion and regrowth included reverse transcription polymerase chain reaction, high speed videomicroscopy, and transcript analysis of novel genetic variants. Results: Three pediatric cases from two families had primary microcephaly from autosomal recessive variants in CEP135 . All cases displayed chronic suppurative respiratory symptoms, recurrent otitis media, and bronchiectasis with low nasal nitric oxide. One case had pathogenic, compound heterozygous, loss of function variants, while the other two cases had homozygous variants of uncertain significance. Transcript analyses of CEP135 variants of uncertain significance supported a splicing defect as disease‐causing. Regrown nasal epithelial cells demonstrated overall normal ciliary ultrastructure with decreased numbers of full‐length axonemes. Ciliary beat pattern was grossly abnormal, and distal axoneme swelling with bulbous‐tip structures were identified pre‐ and post‐cellular regrowth. Conclusion: These cases establish variants in CEP135 as a novel cause of PCD with microcephaly. The TEM findings of bulbous ciliary tips may be a reliable marker of overlapping ciliopathies in patients with aspects of motile and non‐motile ciliary dysfunction.

Background: Nasal nitric oxide (nNO) measurement during velum closure is a useful test for primary ciliary dyskinesia (PCD) but is not feasible in young children. Measurement during tidal breathing is an alternative approach. Our objectives were to define a protocol with age-specific cut-off nNO values during tidal breathing and evaluate this method as an adjunctive test for PCD. Methods: A standardised method for nNO measurement during tidal breathing was developed and tested at the University of North Carolina in children aged −1 at 2 months to 47.7 nL·min ⁻¹ at 5 years of age. Receiver operating characteristic curves had high areas under each curve for nNO cut-offs, ranging from 0.98 to 0.99 across the ages represented in the study. Validation testing at participating sites showed tidal breathing nNO accurately identified 93% of the 60 children aged <6 years with PCD confirmed by ultrastructural ciliary analysis or genetic testing, with high specificity. Conclusion: Tidal breathing nNO measurement is a useful adjunctive test to distinguish young children with PCD from healthy controls as young as 2 months using age-specific cut-off nNO values.

Pathogenic variants in ODAD4 are an ultra-rare cause of primary ciliary dyskinesia (PCD). Previously reported cases display classic disease phenotypes, including chronic oto-sino-pulmonary disease and development of bronchiectasis by adulthood. We report five individuals with PCD harboring biallelic ODAD4 variants (median age 14, range 3–41 years). Participants underwent standardized PCD diagnostic evaluations. Three individuals shared the novel homozygous ODAD4 genotype [NM_031421.5: c.245delA, p.(Lys82Argfs*29)], and genealogy analysis highly suggests a founder effect in French-Canadians from two regions of Quebec. All five participants had normal pulmonary function values. Two Quebec participants lacked radiographic pneumonias or bronchiectasis (ages 14 and 38 years) despite life-long suppurative respiratory symptoms, low nasal nitric oxide levels, and outer dynein arm defects on electron microscopy. Reverse transcription polymerase chain reaction of the c.245delA variant showed abnormal splicing with in-frame skipping of exon 2, allowing expression of a mildly shortened mRNA product. However, functional analysis showed overall static cilia, absence of ODAD4 protein on Western blot, and absence of in vivo mucociliary clearance. The reason for a milder pulmonary phenotype with the c.245delA variant in ODAD4 remains unclear, but regional screening for this variant in Quebec may identify more cases and enhance understanding of this mild form of PCD.

Background: Primary ciliary dyskinesia (PCD) is a rare disease caused by mutations in >50 genes that impair the function of motile cilia. The clinical phenotype is heterogeneous and recent studies have begun to investigate genotype–phenotype relationships to better understand disease pathogenesis and develop improved treatments. The major cause of morbidity and mortality among individuals with PCD is the lack of mucociliary clearance (MCC) that results in chronic respiratory infections and leads to bronchiectasis. Here we examine the relationship between MCC and genotype in two groups of PCD individuals; one with mutations in a gene ( DNAH5 ) that causes PCD with mostly immotile cilia and one with mutations in a gene ( RSPH1 ) that cause PCD with cilia that beat with a near-normal frequency, but an abnormal, sometimes circular waveform. Methods: Patients with known pathogenic variants in DNAH5 (n=8) or RSPH1 (n=7), along with healthy controls (n=8), were assessed for clearance of an inhaled radioactive tracer by mucociliary and cough clearance as measured by gamma scintigraphy. Results: Neither DNAH5 nor RSPH1 subjects showed clear evidence of MCC under either baseline or albuterol stimulated conditions. Unexpectedly, subjects with RSPH1 mutations demonstrated cough clearance (median 9.7%, IQR 6.2–17%) that was significantly higher than subjects with DNAH5 mutations (4.2% (0.94–5.1%); p=0.015) and was not significantly different from healthy control subjects (8.3% (4.2–16%); p=0.88). Conclusions: The results confirm impaired MCC in people with PCD of both genotypes. However, in this small cohort, the results suggest cough clearance may differ between these two genotypes.

Objective: Primary ciliary dyskinesia (PCD) is characterized by upper and lower airway disease. Multiple studies have demonstrated the progression of pulmonary disease; however, longitudinal changes in the otologic and nasal symptoms have not been well described in patients. This study defines age‐related prevalence, age of onset, and age‐related trends in self‐reported otologic and sinonasal comorbidities in individuals with PCD.Study Design: A prospective, longitudinal, multicenter, observational study spanning up to 12 years.Setting: Six PCD centers in North America.Methods: Inclusion criteria were 1 sinus surgery.Conclusion: There is a high prevalence of reported otologic and sinonasal morbidity among people with PCD that begins during early childhood and persists. Further analysis is indicated to evaluate differences over time among participants with varying ultrastructural defects.Level of Evidence: Level 2.

Variation in the non‐coding genome represents an understudied mechanism of disease and it remains challenging to predict if single nucleotide variants, small insertions and deletions, or structural variants in non‐coding genomic regions will be detrimental. Our approach using complementary RNA‐seq and targeted long‐read DNA sequencing can prioritize identification of non‐coding variants that lead to disease via alteration of gene splicing or expression. We have identified a patient with primary ciliary dyskinesia with a pathogenic coding variant on one allele of the SPAG1 gene, while the second allele appears normal by whole exome sequencing despite an autosomal recessive inheritance pattern. RNA sequencing revealed reduced SPAG1 transcript levels and exclusive allele specific expression of the known pathogenic allele, suggesting the presence of a non‐coding variant on the second allele that impacts transcription. Targeted long‐read DNA sequencing identified a heterozygous 3 kilobase deletion of the 5′ untranslated region of SPAG1, overlapping the promoter and first non‐coding exon. This non‐coding deletion was missed by whole exome sequencing and gene‐specific deletion/duplication analysis, highlighting the importance of investigating the non‐coding genome in patients with “missing” disease‐causing variation. This paradigm demonstrates the utility of both RNA and long‐read DNA sequencing in identifying pathogenic non‐coding variants in patients with unexplained genetic disease.

Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous, motile ciliopathy, characterized by neonatal respiratory distress, recurrent upper and lower respiratory tract infections, subfertility, and laterality defects. Diagnosis relies on a combination of tests for confirmation, including nasal nitric oxide (nNO) measurements, high-speed videomicroscopy analysis (HSVMA), immunofluorescent staining, axonemal ultrastructure analysis via transmission electron microscopy (TEM), and genetic testing. Notably, there is no single gold standard confirmatory or exclusionary test. Currently, 54 causative genes involved in cilia assembly, structure, and function have been linked to PCD; this rare disease has a spectrum of clinical manifestations and emerging genotype–phenotype relationships. In this review, we provide an overview of the structure and function of motile cilia, the emerging genetics and pathophysiology of this rare disease, as well as clinical features associated with motile ciliopathies, novel diagnostic tools, and updates on genotype–phenotype relationships in PCD.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type– and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.