The visceral organs of vertebrates have a strikingly conserved left–right (LR) asymmetry of the organ situs that is manifested in the chest (heart and lungs) and abdomen (stomach, spleen, liver, intestine, and colon) (McGrath et al., 2003; Blum et al., 2014). The normal organ asymmetry present across the LR axis of the body is called situs solitus (SS) (Sung et al., 2016). It is well recognized that the leftward flow of extracellular fluid at the node (i.e., nodal flow) plays a major role in normal LR axis determination during embryogenesis (McGrath et al., 2003; Pennekamp et al., 2015). Human LR asymmetry disorders have an estimated probability of more than 1 in 8000 live births and can be divided into two broad classes: situs inversus totalis (SIT) and situs ambiguous (SA). SIT is a malformation featuring a complete mirror image reversal of the organs and is usually not related to major influence on the patient’s health (Levin, 2004; Sung et al., 2016). In contrast, SA, also termed heterotaxy, is defined as any abnormal organ display that was not SS or SIT and is highly associated with human congenital heart disease (CHD) (Zhu et al., 2006; Best et al., 2019; Chen et al., 2020).

Abnormalities in the typical development of laterality usually occur as a result of genetic lesions, which form a number of human heritable disorders with significant clinical implications, including primary ciliary dyskinesia (PCD), nephronophthisis, Carpenter syndrome 2, and male infertility (Olbrich et al., 2002; Otto et al., 2003; Levin, 2004; Twigg et al., 2012; Ding et al., 2020). Variations in the genes related to the development and function of nodal cilia often lead to human laterality defects. Up to now, more than 82 genes have been reported to be related to LR asymmetry disorders, including cilia- and flagella-associated protein family members, coiled-coil domain-containing family members, dynein axonemal assembly factors, dynein axonemal light chains, dynein axonemal intermediate chains, and dynein axonemal heavy chains (DNAHs) (Osório et al., 2019; Al Mutairi et al., 2020; Bustamante-Marin et al., 2020a; Bustamante-Marin et al., 2020b; Cannarella et al., 2020; Chen et al., 2020; Cho et al., 2020; Ding et al., 2020; Heigwer et al., 2020; Sahabian et al., 2020; Sha et al., 2020a; Thomas et al., 2020; Wang et al., 2020; Yang and Qi, 2020; Abdelhamed et al., 2021; Derrick et al., 2021; Guo et al., 2021; Wang et al., 2021). Genes belonging to the DNAH family, such as DNAH1 (OMIM 603332), DNAH5 (OMIM 603335), DNAH6 (OMIM 603336), DNAH9 (OMIM 603330), and DNAH11 (OMIM 603339), are reported to be closely associated with cilia and/or flagella beating (Fliegauf et al., 2005; Hornef et al., 2006; Pifferi et al., 2010; Li et al., 2016). Variations in the dynein axonemal heavy chain 17 gene (DNAH17, OMIM 610063), encoding a component of outer dynein arms (ODAs) in the ciliary axonemes, have been reported to be associated with only flagella destabilization and asthenozoospermia (Whitfield et al., 2019; Zhang et al., 2020). There are, however, comparatively fewer studies that have investigated DNAH17 and multiple morphological abnormalities of the flagella and asthenozoospermia, perhaps limited by the number of LR asymmetry phenotype-associated patients; further research is needed.

In the present study, whole-exome sequencing (WES) combined with Sanger sequencing was used to identify the potential causal gene and variants in two Han-Chinese families with LR asymmetry disorders, and compound heterozygous variants (c.4109C>T and c.9776C>T; c.612C>G and c.8764C>T) in the DNAH17 gene were discovered.

The formation of LR asymmetry of the visceral organs is a conserved feature of human body, which is precisely orchestrated by multiple regulatory mechanisms (Blum et al., 2014; Sung et al., 2016; Hobbs et al., 2018). The process initiating in the node and occurring during embryogenesis can be divided into four steps: i) symmetry breaking caused by the leftward nodal flow, (ii) transmission of asymmetric signals to the left lateral plate mesoderm (LPM), (iii) cascades of Nodal and Lefty2 expression in the left LPM, and (iv) situs-specific morphogenesis (Ramsdell and Yost, 1998; Nonaka et al., 2005; Okada et al., 2005; Yoshimoto et al., 2012).

Rotational movement of motile monocilia in nodal cells creates the nodal flow and activates the asymmetric signaling, while the sperm flagella with similar structure are responsible for cell motility (McGrath et al., 2003; Shiraishi and Ichikawa, 2012; Pennekamp et al., 2015). Most motile cilia and sperm flagella share a highly conserved 9+2 axonemal structure (nine outer microtubule doublets surrounding one central microtubule pair), which are comprised of microtubules, motor dynein arms and the associated structures, exhibiting motile and sensory functions (Zhou et al., 2012; Ishikawa, 2017). Most immotile cilia have a 9+0 axoneme, lacking the central microtubule pair (Fliegauf et al., 2007). The inner and outer dynein arms (IDAs and ODAs), comprised of heavy, intermediate, and light dynein chains, are vital to motility of motile cilia and sperm flagella with 9+2 axonemes (King, 2016; Viswanadha et al., 2017; Lee and Ostrowski, 2021). Human LR asymmetry disorders are thought to be attributed to defective cilia structure or function during embryonic development (Blum et al., 2014; Shinohara and Hamada, 2017). Thus, exploring gene variants targeted establishment and function of nodal cilia during early embryogenesis may help the diagnosis and gene therapy of LR asymmetry disorder.

The DNAH17 gene, located at chromosome 17q25.3, is a large gene composed of 81 exons and encodes an axonemal dynein heavy chain of ODA. DNAHs, also named heavy chains (HCs), include 13 members (DNAH1-3, 5-12, 14, and 17) in humans (Pazour et al., 2006; Inaba, 2011). In the known axial filament complex, the ODAs play a major role in the beating of cilia and flagella through the ATPase activity of their HCs (Whitfield et al., 2019). Dynein HCs are large proteins that turn the energy of ATP into force supporting the sliding of outer microtubule doublets, which generates the beating of cilia (Pazour et al., 2006). To date, variants in most genes of DNAHs in humans have been reported to be associated with diseases related to cilia or flagella. A common autosomal recessive disorder caused by those variants is PCD, which is characterized by recurrent respiratory tract infections, laterality defects, and/or infertility, with highly genetic and clinical heterogeneity (Fliegauf et al., 2005; Hornef et al., 2006; Pifferi et al., 2010; Li et al., 2016). In the ultrastructure, sperm flagella are similar to cilia, underpinning the common relationship between male infertility with PCD and subfertility in women with PCD due to deficient ciliary function in the oviducts (Lucas et al., 2014). Of interest, variants in DNAH1 and DNAH9 genes, reported to be associated with PCD, have been depicted in patients with only male infertility, resulted from asthenozoospermia, without other ciliary disorders (Ben Khelifa et al., 2014; Fassad et al., 2018).

In this study, the proband from family 1 presented with SIT without any other cilia-related symptoms, and compound heterozygous variants c.4109C>T (p.Thr1370Ile) and c.9776C>T (p.Ala3259Val) in the DNAH17 gene were identified using a combination of WES and Sanger sequencing. The second DNAH17 compound heterozygous variants, c.612C>G (p.Ile204Met) and c.8764C>T (p.Arg2922Cys), were found in the proband from family 2. The boy presented with dextrocardia and CHD. Cardiac murmurs with cyanosis and recurrent cough were discovered in infancy, suggesting that he may suffer with the cilia-related symptoms. Detailed clinical characteristics of the available family members with DNAH17 variants are presented in Table 3. It seems that at least 10% (2/20) DNAH17 compound heterozygous or homozygous carriers have LR asymmetry disorders. There are only a few studies on DNAH17, and none was found on studying the gene function due to the large size of DNAH17 molecular mass (510 kDa). In addition, the current knowledge on the crucial part of DNAH17 playing in flagella destabilization and asthenozoospermia may depend on genetic or environmental factors such as the mutation type, organism or context (Whitfield et al., 2019; Zhang et al., 2020). Actually, different DNAHs members have been shown to play an important role in cilia/flagella formation and cilia/flagella regulation. DNAH1 and DNAH9, homologs of DNAH17, were located to ciliary axonemes and sperm flagella, and responsible for cilia/flagella-related phenotypes (Bartoloni et al., 2002; Olbrich et al., 2002; Ben Khelifa et al., 2014; Fassad et al., 2018). Due to a common highly conserved 9+2 axonemal structure of cilia and sperm flagella, different DNAH17 mutations may independently cause flagella destabilization, asthenozoospermia or LR asymmetry disorders in specific organelle sharing a common axonemal machinery. Though DNAH17 expression was detected in testis, brain, lung and other tissues (https://www.ncbi.nlm.nih.gov/gene/8632), the protein was only observed in the flagella by assays of immunoblotting and immunofluorescence staining in various human somatic cell lines, human respiratory epithelial cells and sperm cells (Fagerberg et al., 2014; Whitfield et al., 2019; Zhang et al., 2020). It implies that DNAH17 expression may be influenced by cell type-specific spatial localization and the switch point in the development of the nodal flow during early embryogenesis. The lack of typical symptoms, such as nasosinusitis and bronchiectasis, in the two patients may be due to absent or low expression of DNAH17 in specific tissues after the completion of the embryonic development. Biallelic variant types of DNAH17, genetic background, and epigenetic modification, as well as environmental factors, may potentially affect the phenotypic manifestation. The possible genotype-phenotype association should be warranted with more DNAH17-mutated carriers discovered. Our observation of the potential relationship between DNAH17 and LR asymmetry disorders may extend the field-of-view for new actor of DNAH17 in the development of human diseases.

Taken together, our research identified compound heterozygous DNAH17 variants (c.4109C>T and c.9776C>T; c.612C>G and c.8764C>T) in families with LR asymmetry disorders, typical phenotypes of ciliary disorders, including SIT, dextrocardia, and CHD, albeit infertility cannot be excluded. To our knowledge, this is, the first report of relationships between DNAH17 variants and ciliogenesis, which expands the phenotypic spectrum and benefits genetic counseling. Combined with the reported DNAH17-associated asthenozoospermia, we proposed that DNAH17 compound heterozygous variants, or homozygous variants, may potentially cause a specific disease, the DNAH17-associated ciliary/flagellar disorder. The study may be limited by the lack of nasal epithelial brush biopsy samples for ciliary beating and ultrastructure analysis. Further constructing DNAH17 variant-targeted animal models and performing experimental therapies will facilitate an in-depth comprehension of cellular and molecular mechanisms of ciliary and flagellar defects, and contribute to rectification of the defects.