Submission Details

Primary Ciliary Dyskinesia (PCD) is a genetically heterogeneous group of disorders, usually beginning in early childhood, characterized by chronic cough, recurrent infections of the upper and lower respiratory tract, randomization of left/right body asymmetry, and subfertility (PMID: 32943623). A subset of PCD cases have complex congenital heart defects (PMID: 17515466). The PCD phenotype results from structural and/or functional abnormalities of motile cilia and flagella. Ciliary motility is powered by a set of axoneme-specific dynein motor complexes. Mutations in genes encoding proteins involved in the assembly, transport, or docking of dynein motor complexes can result in ciliary immotility or dysfunction (PMID: 31624012). Outer dynein arm (ODA) motor complexes are produced and preassembled in the cytosol, transported to the ciliary or flagellar compartment, and then anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. The ODA-DC facilitates binding and may also play a role in regulating the activity of the ODAs (PMID: 11907279). The vertebrate ODA-DC is comprised of 5 subunits: ODAD1 (CCDC114), ODAD2 (ARMC4), ODAD3 (CCDC151), ODAD4 (TTC25), and ODAD5 (CLXN) (PMID: 34715025). Defects in each of these subunits have been associated with PCD (PMIDs: 23261303, 238497783, 25192045, 27486780, 36727596).

The CLXN (Calaxin) gene, also known as ODAD5 (Outer Dynein Arm Docking Complex subunit 5) or EFCAB1 (EF-hand calcium binding domain 1), was first reported in relation to PCD patients by Hjeij et al. in 2023 (PMID: 36727596). Hjeij et al. describe two independent patients with loss-of-function CLXN mutations that present with laterality defects and chronic respiratory symptoms. One of these patients also had a complex congenital cardiac malformation. A third fetal patient with complex congenital heart defects was not viable and could not be assessed for respiratory symptoms. Two of these patients also presented with neurologic developmental abnormalities. It is unclear if these phenotypes were associated with pathogenic CLXN variants (related to cardiac abnormalities or defects in brain ependymal cilia) or unrelated to PCD.

The specific disease entity associated with CLXN is Primary Ciliary Dyskinesia 53, an autosomal recessive disorder caused by biallelic mutation in the CLXN gene. Per criteria outlined by the ClinGen Lumping and Splitting Working Group, we found the molecular mechanism and autosomal recessive mode of inheritance to be consistent among unrelated patients, while the phenotypic variability among them appeared to represent a spectrum of disease rather than separate disease entities. Therefore, cases caused by inherited CLXN variants have been lumped into a single disease entity, Primary Ciliary Dyskinesia 53 (MONDO:0957991, OMIM # 620642).

Genetic evidence from three probands carrying biallelic loss-of-function CLXN mutations in one publication was evaluated in this curation (PMID: 36727596). Two unique homozygous variants from two probands were scored (both nonsense mutations). A third homozygous frameshift variant from a fetal patient was not scored in this curation, since the patient could not be assessed for respiratory phenotype, a hallmark of PCD. An additional unpublished proband with homozygous loss-of function CLXN variants and a phenotype consistent with published probands has also been identified (M. Lejendre, personal communication, February 13, 2024).

The gene-disease association between CLXN and PCD 53 is supported by gene expression data that show CLXN expression in ciliated tissues. GTEx RNA-seq expression studies in reference human tissues show strong expression of CLXN in lung, testis, brain, and Fallopian tube (PMID: 23715323). This pattern is consistent with CLXN as a subunit of the ODA-DC in motile cilia and flagella (sperm). Murine Clxn is expressed at the mouse left-right organizer (LRO) during early embryogenesis. Laterality defects linked to CLXN mutations in humans likely result from dysfunctional LRO monocilia (PMID: 31286071).

CLXN is part of the five subunit ODA-DC that stabilizes the docking of ODAs onto ciliary doublet microtubules (PMID: 37057896). Immunofluorescence microscopy studies were performed on respiratory epithelial cells from control and affected individuals carrying biallelic pathogenic variants in ODA-DC subunit genes ODAD1, ODAD2, ODAD3, and ODAD4. CLXN was not detected along the ciliary axonemes of cells with any of these ODA-DC defects, confirming that CLXN associates with the other subunits to form the ODA-DC in humans. In contrast, CLXN is present in the axonemes of control cells and also those carrying DNAH5, DNAI1, DNAI2, or DNAH9 mutants (encoding ODA proteins), indicating that CLXN is not a component of ODA complexes in human respiratory cilia (PMID: 36727596). TEM cross section studies on respiratory epithelial cells from CLXN-PCD 53 patients show a loss of ODAs from the distal axonemes. Immunofluorescence studies on patient cells confirm the absence or mislocalization of ODA heavy chains, suggesting that in the absence of CLXN, there is a failure to correctly assemble/dock the distal ODAs.

CLXN is a calcium sensor protein with three EF-hand Ca2+-binding domains and it has been suggested that in addition to helping in the stabilization of ODAs onto the axoneme, it may play a role in calcium-mediated regulation of ciliary beating (PMIDs: 36727596, 38876797). CLXN was first described in the sperm flagella of Ciona intestinalis as a calcium dependent modulator of ODAs, regulating wave propagation and swimming direction (PMID: 18620543). Inhibition of calcium dependent CLXN with repaglinide (a neuronal calcium sensor family protein inhibitor) in human and ferret tracheal tissue alters the dynein motor protein function of cilia on the airway surface, leading to an impaired wave of metachrony (coordinated asymmetric ciliary movement) across the surface, reducing mucociliary transport (PMID: 38860289). Mucociliary clearance via the coordinated movement of cilia is critical for maintaining lung health. An alteration in the function of CLXN that leads to a reduction in mucociliary clearance is consistent with the respiratory phenotypes seen in PCD with CLXN mutations.

Two non-human experimental models have been used to support the gene-disease relationship between CLXN and PCD 53. RNAi-based knockdown of clxn (efcab1) transcripts in the planarian Schmidtea mediterranea reduces the velocity of locomotion, supporting a reduction or change in ciliary motility. Clxn (Efcab1) knockout mice display phenotypes consistent with human PCD, including hydrocephalus, situs inversus, and abnormal motility of tracheal cilia, ependymal cilia, and sperm flagella (PMID: 31286071). In addition, the formation of embryonic nodal (LRO) monocilia is disrupted in these knockout mice (sparce and dysmotile nodal cilia). Motility of nodal cilia in the embryonic LRO is necessary for the determination of correct left–right body asymmetry. Fluorescent microbead studies show that there is reduced flow velocity and random flow at the embryonic node in Clxn knockout mice embryos, consistent with the laterality defects seen in mice as well as with situs abnormalities and cardiac defects seen in human CLXN-PCD 53 patients. Clxn protein expression and PCD phenotypes can be rescued in knockout mice upon reintroduction of wild-type murine Clxn gene on a bacterial artificial chromosome. (PMID: 31286071).

In summary, there is strong evidence supporting a gene-disease relationship between variants in CLXN and Primary Ciliary Dyskinesia 53. This classification was approved by the ClinGen Motile Ciliopathy GCEP on March 3, 2025 (SOP Version 11).