Submission Details

DNAAF4 (also known as DYX1C1) was first reported in relation to autosomal recessive primary ciliary dyskinesia 25 (PCD25) in 2013 (Tarkar et al., PMID: 23872636). PCD25 is one of nearly 50 primary ciliary dyskinesias observed to be inherited in a monogenic fashion. Most PCD patients present with a chronic productive cough, recurrent respiratory infection, and chronic rhinosinusitis. Some patients also demonstrate situs inversus and neonatal respiratory distress (PMID: 33242470). Transmission electron microscopy (TEM) analysis of the respiratory cilia from all of the examined probands included in this curation demonstrated combined outer and inner dynein arm defects (PMIDs: 23872636, 35903363).

Variants in DNAAF4 have also been observed in association with susceptibility to dyslexia (PMID: 12954984). Multiple studies surrounding this association have yielded conflicting results (PMID:15520411, PMID: 15702132). Per criteria outlined by the ClinGen Lumping & Splitting Working Group, we found differences in molecular mechanism, inheritance pattern, and phenotypic variability relative to primary ciliary dyskinesia. Therefore, cases diagnosed with susceptibility to dyslexia or primary ciliary dyskinesia have been split into separate DNAAF4 curations. This curation will focus only on primary ciliary dyskinesia 25 (OMIM:615482).

This curation includes eleven variants (eight nonsense, one frameshift, one consensus splice-site, and one large deletion) that have been reported in twelve probands in two publications (PMIDs: 23872636, 35903363). A deletion of 3549-bps encompassing exon 7 of DNAAF4 was seen in six apparently unrelated probands (four heterozygotes and two homozygotes, PMID: 23872636)]. Of these six probands, one homozygote was from a highly consanguineous family of Irish descent, and one compound heterozygote was from a family of Australian ancestry. Three other compound heterozygotes and one homozygote were from families from North America. The 3549-bp deletion was observed in compound heterozygosity with four separate apparent loss-of-function variants (p.Glu109Ter, p.Arg270Ter, p.Thr85fs, and c.783+1G>T) in four independent families (PMID:23872636). The c.583delA (p.Ile195Ter) variant was also observed in 2 apparently unrelated homozygous probands of German descent (PMID: 23872636). More cases were available in the literature, but were not included in the curation as the maximum score for genetic evidence (12 pts) had been reached. The mechanism of pathogenicity appears to be loss-of-function.

This gene-disease relationship is also supported by experimental evidence including expression studies, functional assays, protein interaction evidence, and animal models (PMIDs: 23715323, 19944400, 23872636, 23872636, 23872636). GTEx expression data revealed high levels of DNAAF4 in the brain, fallopian tube, lung, testis, and thyroid compared to other tissues in unaffected individuals, which is consistent with the PCD phenotype (PMID:23715323). Interaction evidence from a co-immunoprecipitation experiment demonstrated interaction between Dnaaf4 and Dnaaf2 (PMID:23872636). Since DNAAF2 was definitively associated with PCD10 according to the Motile Ciliopathy GCEP, the interaction of the 2 proteins provides further evidence for the association between DNAAF4 and PCD. Immunofluorescence in patient cells showed that both DNAAF4 and DNAAF1 play a role in the assembly and recruitment of other proteins (such as DNAH9, DNAH5, DNAI2, and DNALI1) to the cilia. Since the association between DNAAF1 and PCD13 was previously classified as definitive by the Motile Ciliopathy GCEP, the similar molecular defects in cells from both DNAAF1 and DNAAF4 patients support the suspected role of DNAAF4 variants in contributing to the PCD phenotype (PMIDs: 23872636, 19944400). A Dnaaf4 knockout mouse model demonstrated laterality defects (59% situs inversus totalis) and combined outer and inner dynein arm defects, similarly to human patients with PCD25. The Dnaaf4 KO mice also demonstrated immotile cilia in cerebral ventricles (PMID: 23872636). Finally, a zebrafish model created using morpholinos recapitulated the laterality defects (37.9% SIT, 25% heterotaxia), and immotile cilia in Kupffer's vesicles, which are consistent with phenotypes seen in PCD25 patients (PMID: 23872636).

In summary, DNAAF4 is definitively associated with autosomal recessive primary ciliary dyskinesia 25. This has been repeatedly demonstrated in both the research and clinical diagnostic settings, and has been upheld over time. This classification was approved by the ClinGen Motile CIliopathy Gene Curation Expert Panel on the meeting date April 11th, 2024 (SOP Version 10).