Submission Details

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous pathology affecting mostly the respiratory tract. Laterality defects and infertility are also reported in PCD patients, due to structural similarities between tracheal cilia, nodal cilia, fallopian tube cilia and sperm flagella. Genes encoding ciliary components and ciliogenesis factors are PCD candidates. DNAH9 encodes a constituent of the outer dynein arms (ODA) located in the distal part of airway cilia. Mutations in this gene were first described in 2018 in patients with laterality defects and mild or no respiratory signs (PMID: 30471717 and PMID: 30471718). Between 2018 and 2024, 16 different papers reported DNAH9 variants in a total of 31 patients. A total of 50 different mutations were detected in the reported patients and consisted of 22 null mutations, 1 in-frame deletion and 27 missense variants (PMID: 30471717; 30471718; 33139725; 35202559; 32253119; 33182294; 32037394; 33452233; 33610189; 35050399; 35116053; 35770021; 36003331; 36747106; 32996775; 36712782; 39115416).

This gene curation has evaluated the gene-disease relationship between DNAH9 and primary ciliary dyskinesia 40 (MONDO:0032664). While most of the patients with DNAH9 defects had laterality defects and mild or no respiratory signs, the Motile Ciliopathy GCEP defines a primary ciliary dyskinesia phenotype as including a chronic wet cough alongside either chronic year-round nasal congestion from birth or early childhood, unexplained neonatal respiratory distress in a term infant, or an organ laterality defect. Patients who did not meet these phenotypic criteria had their variants downscored to zero for the purposes of this curation.

Laterality defects were observed in 20/31 (65%) patients and asthenozoospermia was reported in 4 male patients. Nasal nitric oxide measurements in 13 patients with DNAH9 mutations showed normal to high values (36-220 nL/min) (PMID: 30471717; 30471718; 33139725; 33452233; 35050399; 32996775). Taking all patients into account, the genetic evidence score reached a maximum of 12 points. Ciliary investigations by transmission electron microscopy showed partial absence of the outer dynein arms. Electron tomography showed more precise ciliary abnormality consisting of a reduced volume of ODA from the distal cilia (PMID: 30471717). This is consistent with immunofluorescence assays showing absence of DNAH9 and distal absence of DNAH5, DNAI1, DNAI2 and DNALI1 in respiratory cilia (PMID: 30471717; 30471718). A reduced ciliary distal bending was observed with high speed video microscopy (PMID: 30471717; 30471718).

The DNAH9 protein is a dynein axonemal heavy chain composed of 4486 amino acids. This protein is expressed in lungs and in fallopian tubes (PMID: 25613900). Immunofluorescence staining showed its expressions in respiratory cilia and its absence from sperm flagella. Patients with DNAH9 mutations showed altered expression of the heavy chain from cilia (PMID: 30471717; 30471718). Moreover, the cilia from PCD patients with biallelic variants in DNAAF1 or DNAAF3 showed altered expression of DNAH9 in the distal part of the ciliar. (PMID: 22387996 and 19944400). The interaction between DNAH9 and CCDC114 (ODAD1); another protein with genetic variants associated with PCD, was demonstrated through co-immunoprecipitation (PMID: 30471718). The knock-down of DNAH9 in paramecium through RNAi leads to loss of ciliary ODA, reduced ciliary beat frequency and an abnormal motility pattern (PMID: 30471717). Zebrafish embryos with dnah9 knockdown using morpholino-oligos show laterality defects and hydrocephalus, but no disturbance of ciliary number or length in the retina or Kupffer’s vesicle (PMID: 35050399). Two mouse models were generated with the CRISPR/Cas9 system leading to the deletion of exons 2-4 (PMID: 35050399; 35729109). The first model showed hydrocephalus and compromised cardiac function, ultrastructural defects in outer dynein arms, limited range of ciliary bending, and insufficient effective stroke, but no laterality defects (PMID: 35050399). Mice from the second model showed absence of distal ciliary ODA and a reduced ciliary beat frequency (PMID: 35729109). Experimental evidence including expression pattern, protein interaction and non-human models; reached the maximum of 6pts.

In summary, DNAH9 is definitively associated with autosomal recessive primary ciliary dyskinesia 40. This has been repeatedly demonstrated in both the research and clinical diagnostic settings, and has been upheld over time without the emergence of conflicting evidence. This classification was approved by the ClinGen Motile Ciliopathy GCEP on September 19th, 2024 (SOP Version 10).