Submission Details

Visceral heterotaxy belongs to a genetically and clinically heterogeneous set of syndromes characterized by abnormalities of the lateralization of internal organs and midline structures, resulting from failure to establish normal left-right asymmetry during embryogenesis. In addition to abnormal symmetry and malposition of the thoraco-abdominal organs and vessels, heterotaxy can include complex congenital heart defects and extra cardiac manifestations (PMIDs: 16113522, 33603285). Ciliated cells at the embryonic node generate a leftward flow of extra-embryonic fluid that is essential for left-right determination (PMID: 20066075). Dysfunction in the nodal cell cilia disrupts the “nodal flow” and can result in laterality defects. Heterotaxy syndrome can occur in isolation or can be a feature of other genetic syndromes such as the motile ciliopathy, primary ciliary dyskinesia (PCD). PCD phenotypes result from structural and/or functional abnormalities of motile cilia and flagella. Ciliary motility is powered by a set of axoneme-specific dynein motor complexes. The cytoplasmic assembly of these large multiprotein complexes is assisted by dynein axonemal assembly factors and their subsequent transport into cilia is facilitated by proteins involved in the intraflagellar transport (IFT) system.

Variants in genes encoding proteins involved in the assembly or transport of these complexes can result in the improper assembly of dynein chains or impaired transport of complexes into the axoneme, causing defects in outer dynein arms (ODA) and/or inner dynein arms (IDAs) that can result in ciliary immotility or dysfunction (PMID: 31624012). DAW1 (Dynein Assembly Factor with WD Repeats 1), also referred to as ODA16 and WDR69, encodes a transport adaptor for the multi-subunit IFT-B1 complex, facilitating the efficient transport of ODAs along the axoneme (PMIDs: 18852297, 30133350).

DAW1 was first reported in relation to heterotaxy and congenital heart disease in 2017 (Jin et al., PMID: 28991257). Two probands with heterotaxy and cardiac phenotypes, both of whom carried biallelic DAW1 variants, were reported. No respiratory symptoms suggestive of a PCD diagnosis were described for these patients. In 2022, Leslie et al. described a motile ciliopathy characterized by laterality defects caused by biallelic variants in DAW1 in two unrelated families with an autosomal recessive mode of inheritance (PMID: 36074124). The first family had phenotypes suggestive of a mild form of PCD, including a laterality defect (situs inversus) and oto-sinopulmonary phenotypes, whlie the second family had laterality and complex congenital heart defects but no respiratory symptoms.

Upon review of the genetic evidence available for DAW1, the ClinGen Motile Ciliopathy GCEP concluded that the molecular mechanism (biallelic loss of function) and the mode of inheritance (autosomal recessive) are consistent among unrelated patients carrying DAW1 variants. However, the variability in respiratory phenotypes reported among patients was notable and suggestive of reduced evidence of a DAW1 relationship to PCD as opposed to an isolated laterality defect. PCD is a motile ciliopathy with a respiratory phenotype. Therefore, per criteria outlined by the ClinGen Lumping & Splitting Working Group, DAW1 was recommended for split curation for two distinct disease assertions; primary ciliary dyskinesia 52 (MONDO: 0957922, OMIM # 620570) and visceral heterotaxy (MONDO: 0018677). This curation has evaluated all of the available genetic evidence for DAW1, however, scoring has only been reported for cases relevant to a gene-disease association between DAW1 and visceral heterotaxy. Evidence from 3 probands in 2 publications has been included in this curation, representing 4 unique variants (2 missense, 2 nonsense), (PMIDs: 28991257, 36074124). The mechanism of pathogenicity for DAW1 variants is biallelic loss-of-function.

The gene-disease association between DAW1 and PCD 52 is supported by gene expression data that shows high DAW1 levels in ciliated tissues such as lung, testis, and fallopian tube (PMID: 23715323), consistent with the proposed role of DAW1 in the transport of ODAs in motile cilia and flagella (PMID: 32943623). RNA in situ hybridization studies of embryonic mice show that Daw1 is expressed in the embryonic node (PMID: 36074124), consistent with Daw1's role in left-right organization. The efficient transport of ODAs into cilia is mediated by the interaction of DAW1 with both ODAs and the IFT46 protein of the multi-subunit IFT-B1 complex (PMID: 18852297, 30133350). DAW1 plays a role as a linker protein between these complexes. IFT46 is a core component of the IFT machinery and is required for the formation of all cilia. In Xenopus, knockdown of IFT46 results in a shorter body axis as well as the formation of fewer and shorter cilia (PMIDs: 27320864, 33628615). Yeast two-hybrid experiments confirm an in vivo interaction between ODA16 (human) and IFT46 (mouse) (PMID: 18852297). Co-immunoprecipitation studies in Chlamydomonas confirm that ODA16 physically interacts with ODAs (PMID: 28298440). RNAi knockdown of daw1 in the planarian Schmidtea mediterranea resulted in locomotion defects and protonephridial dysfunction (edema) (PMID: 32359074). These phenotypes are indicative of ciliary dysfunction in cells functionally dependent on motile cilia: the planarian epidermis and the protonephridial excretory system. Zebrafish wdr69 antisense morpholino knockdown produced embryos with phenotypes consistent with defective cilia in multiple tissues: curled tail, pronephric cysts, pericardial edema, and extra otoliths (PMID: 20568242). Embryos were shown to have defective ciliary motility in the Kupffer’s vesicle and developed left-right asymmetries in the heart and gut. Electron microscopy of Kupffer’s vesicle cilia showed an absence of ODAs, confirming that Wdr69 (DAW1) is needed for ODA assembly in the embryonic axoneme. Mice homozygous for a Wdr69 loss-of-function missense variant were studied as a mammalian PCD model (PMID: 28289722). The wdr69–/– mouse model recapitulates phenotypes one might associate with a PCD patient with ODA defects. Wdr69–/– mice respiratory cells show an absence or reduction in ODAs as well as dyskinetic ciliary motion, despite normal ciliated area and ciliary beat frequency. The absence of ODAs is consistent with the role DAW1 is thought to play in the transport of ODAs along the axoneme. These mice have impaired mucociliary clearance and laterality defects such as situs inversus and heterotaxy with complex congenital heart defects. Most of these mice die before term from complex congenital heart defects or soon after birth from hydrocephalus.

In summary, there is moderate evidence supporting a gene-disease relationship between DAW1 and visceral heterotaxy. This association has been repeatedly demonstrated in both research and diagnostic settings, and has been upheld over time without the emergence of contradictory evidence. This classification was approved by the ClinGen Motile Ciliopathy GCEP on July 11, 2024 (SOP Version 10).