Novel Pathogenic DNAH5 Variants in Primary Ciliary Dyskinesia: Association with Visceral Heterotaxia and Neonatal Cholestasis

Abstract

The dynein axonemal heavy chain 5 gene codes for a subunit of axonemal dynein necessary for ciliary motor function. Though research has elucidated the consequences of some variants in this gene, it is still unclear whether many variants in the DNAH5 locus are benign or pathogenic due to the rarity of primary ciliary dyskinesia (PCD, of which Kartagener's syndrome is a subset). Here, we introduce the case of an infant boy presenting with the classical findings of PCD along with visceral heterotaxia and neonatal cholestasis. Genetic testing indicated that the patient is a compound heterozygote with a pathogenic c.8498G > A (known as pathogenic) on the maternally derived allele and two variants of uncertain significance, c.1206T > A and c.7800T > G, on the paternally derived allele. As PCD is autosomal recessive, we conclude that one, or both, of these paternally derived variants are pathogenic. To our knowledge, this is the first time that the clinical implications of c.1206T > A (p.Asn402Lys) and c.7800T > G (p.Ile2600Met) are documented. Furthermore, we use this case as an example to recommend clinicians to assess for PCD and laterality defects when presented with severe infantile cholestasis. While the association of cholestasis with PCD is relatively uncommon, PCD is a risk factor for increased prevalence of biliary atresia and infections, both of which are known causes of cholestasis in early infancy.

Introduction

Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder with an estimated prevalence of 0.01% (1/10,000) to 0.005% (1/20,000) in live births, though actual ratios are likely higher due to unreported or unidentified cases. ¹ Approximately, 90% of PCD presentations result from dynein defects, of which approximately 38% involve variants in dynein axonemal intermediate chain 1 ( DNAI1 ) and dynein axonemal heavy chain 5 ( DNAH5 ). ² DNAH5 variants are found in 28% of patients with outer dynein arm (ODA) defect and in 53% of patients with PCD, ³ making this a key protein of interest in ciliopathies. As shown in Fig. 1 , ODA complexes are attached to the outer ring of microtubules and each consists of 3 heavy chains, 2 intermediate chains, and 11 light chains. ⁴ As one of the three heavy chains, DNAH5 consists of 79 exons and is required for proper microtubule sliding and ciliary motion. ⁵ Dynein is a ciliary microtubule-associated motor protein that aids in microtubule movement and molecular transport. It utilizes adenosine triphosphate to ambulate toward the minus (−) end of microtubules and is also involved in early stages of mammalian mitotic spindle formation, ⁶ viral replication, ⁷ and eukaryotic cilium function.

Fig. 1.

Simplified schematic of motile 9 + 2, motile 9 + 0, and immotile 9 + 0 cilia ultrastructure (adapted from Tarkar 2015 ⁸ ).

Background

Cilia are finger-like cellular protrusions constituted of microtubules and associated proteins such as dynein. There are three general classes of cilia demarcated by function: motile 9 + 2, motile 9 + 0, and nonmotile 9 + 0. Motile 9 + 2 cilia are found in locales such as on the apical surfaces of the respiratory tract, ependymal cells of the CNS, and the fallopian tube oviducts. They have axoneme consisting of nine outer microtubule pairs and one central pair. ⁸ Radial spokes (shown in Fig. 1 ) are multisubunit proteins with a postulated function of synchronizing displacement, allowing for whip-like movement ⁹ and the resultant transport of extracellular material. Two types of defects may afflict PCD patients due to motile 9 + 2 ciliopathy: motor and sensory. In the former category, physical movement and the mucociliary escalator fails, resulting in increased infections of the respiratory tract, ear, and lungs. Patients may be rendered infertile due to defective sperm flagella in males and defective oviducts in females. Research has shown that motile cilia can also have chemosensory functions in several tissue types; from respiratory cilia that detect bitter, noxious substances ¹⁰ to cilia defects in airways that cause bronchiectasis. ¹¹

Motile 9 + 0, or nodal cilia, lack the central microtubule pair and are only present in the ventral node of the gastrula during embryonic development. Its uncoordinated whirling motion is known to be a major determinant of left-right symmetry ³ ; as such defects in nodal cilia may result in situs inversus and heterotaxia disorders.

Nonmotile 9 + 0 cilia are also known as primary cilia. A point of confusion may result from semantics; contrary to the naming, primary cilia are not the subtype associated with PCD as they do not possess ODAs—though they do utilize cytoplasmic dynein and kinesin for molecular transport. ¹² Nonmotile 9 + 0 cilia are thus the only type that are not affected by DNAH5 ciliopathy. These are found on renal tubules, bile ducts/cholangiocytes, bone, cartilage, retina, cochlea, developing heart, and fibroblasts. ^{13 14} They detect and modulate bile as it passes through the biliary tree, ^{13 15} perform chemo-, osmo-, and mechano-sensation in various organs, and have been found to be involved in cellular signaling. ^{10 12}

Clinical Manifestations of Primary Ciliary Dyskinesia

The clinical presentation of PCD may result from the motile 9 + 2 cilia defects given above. Lack of functional motile 9 + 2 cilia commonly results in recurrent respiratory infections, mucosal infections, and infertility. ³ As mentioned, motile 9 + 2 cilia are also found in CNS ependymal cells; thus, in rare circumstances, hydrocephalus may develop as a result of impaired cerebrospinal fluid circulation. ^{16 17} Approximately half of PCD patients (Kartagener's heterotaxia subset) also present with motile 9 + 0 cilia defects that result in situs inversus, heterotaxia, and abnormal organ localization. Though relatively uncommon, PCD is also associated with neonatal cholestasis via increased likelihood of bacterial or viral infections ¹⁸ and biliary atresia owing to midline or transverse liver positioning ¹⁹ as seen in the visceral heterotaxia subset.

Diagnosis

Current guidelines recommend that all children with recurrent nasal/respiratory infections, bronchiectasis, cystic fibrosis, refractory chronic cough, and laterality defects be tested for PCD. These tests include ultrastructure analysis (gold standard), genetic testing for variants in cilia genes (e.g., DNAH5 and DNAI1 ), high speed microscopy of cilia function, nasal nitric oxide, or immunofluorescence imaging of antibody labeled DNAH5 . ²⁰ To gauge the pretest likelihood of PCD clinically, clinicians may use Primary Ciliary Dyskinesia Rule, a screening tool that offers a relatively high sensitivity (0.90) and specificity (0.75) for scores ≥5. ²¹

Management

Management of PCD is twofold. The first is to correct emergent congenital abnormalities that may be life threatening (e.g., right-to-left heart shunts). The second and the long-term strategy focuses on addressing mucosal and respiratory tract infections. Due to defective mucociliary ladder and impaired clearance of mucus, positive expiratory devices, vest therapy, and active cycle breathing techniques may be helpful. ²² Infections should be treated promptly, and antibiotic choice should be decided with assistance of sputum or bronchoalveolar lavage cultures (e.g., tobramycin for Pseudomonas aeruginosa ). Due to augmented concern for pulmonary/respiratory tract infections and bronchiectasis, patients should stay updated on vaccinations, particularly for influenza and Streptococcus pneumoniae . ²³ While not ubiquitously used presently, recent multinational trials have shown that maintenance azithromycin therapy is safe and may significantly reduce the rate of respiratory infections and exacerbations, ²⁴ though long-term efficacy is not yet known and there are concerns for the development of azithromycin-resistant bacteria. ²⁵

Prognosis

PCD presents with significant morbidity with varied clinical manifestations as mentioned above. Long-term mortality prognosis depends on severity and frequency of respiratory infections as well as access to prompt medical care. In limited cases, lung transplant may become necessary later in life. ¹ It is important to note that PCD diagnoses are often delayed or missed. Numerous study results show that early detection and treatment of PCD have been found to reduce long-term pulmonary morbidity and improve disease course. ^{26 27}

Case Presentation

Video 1 High magnification oil immersion microscopy depicting shortened cilia with restricted, asynchronous motility (locale 1).

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Video 2 High magnification oil immersion microscopy depicting shortened cilia with restricted, asynchronous motility (locale 2).

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Video 3 High magnification oil immersion microscopy depicting shortened cilia with restricted, asynchronous motility (locale 3).

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The patient is a 31-month-old male born at term with a complicated neonatal course. He had double outlet right ventricle, twin atrioventricular nodes, patent ductus arteriosus (PDA), total anomalous pulmonary venous return (TAPVR), hypoxemia, tachypnea, tachycardia, and heterotaxia with asplenia, preaxial polydactyly, midline liver, right upper quadrant stomach, and malposition of great arteries. Notably, there was no evidence of maternal diabetes mellitus, renal issues, adverse environmental exposures, or familial history of birth defects. Using the PICADAR scoring system, this patient scored a 10 (respiratory symptoms, neonatal unit, abnormal situs, and congenital heart defect), suggesting a 92.6% pretest probability of PCD. Subsequent TAPVR repair, PDA ligation, and tunnel repair procedures were conducted, and pre-axial polydactyly was treated by surgical excision. As a neonate, the patient had feeding issues, stunted growth, progressive hyperbilirubinemia, and acholic stools. The gastroenterology team suspected biliary atresia. Subsequent liver biopsy was interpreted as cholestatic ductular reaction with nonuniform periductulitis but lacked bile duct proliferation and was not typical for large duct obstruction. Intraoperative cholangiography at the age of 2 months showed patent extrahepatic bile ducts with narrowed openings. Histology was unchanged but generalized paucity of the interlobular bile ducts suggests maturation delay that resembled the ductal plate stage of small duct development ( Fig. 2A ). Multiple small intrahepatic bile ductules from the liver wedge biopsy were examined with EM imaging. Ultrastructure showed numerous cholangiocytes with fragmented and disorganized plasma membranes ( Fig. 2B ). Interestingly, one unique 9 + 0 primary cilium was found to have peculiar structural change resembling “7 + 2” morphology ( Fig. 2C ). According to international guidelines, had this disorganization been seen in ≥25% of at least 50 well-oriented motile 9 + 2 cilia with inner dynein arms (IDA) defect in respiratory cilia (CCDC39–40 mutations, and absence of N-DRC proteins which include DNALI1), it would suggest class 1 defect or diagnosis of PCD. If seen consistently in well-oriented respiratory cilia with IDA present (mutations associated with genetic variants in cyclin O and multicilin), it would suggest Class 2 defect (PCD diagnosis with supporting evidence). ²⁸ However, unlike motile cilia, consensus guidelines are lacking for nonmotile cilia. Thus, the significance of one abnormal cholangiocyte cilium with unusual singlet-doublet microtubule structure in the presence of mostly normal 9 + 0 cilia ( Fig. 2C ) is unclear. However, the preponderance of mostly normal 9 + 0 primary cilia suggests that the aforementioned changes are unlikely to be the result of genetic mutation. While neither precedent nor etiology for this microtubule structural change has been reported in literature, the dysplasia and fragmentation could be the result of ductal plate-like maturation delay and/or damage caused by accumulation of cytotoxic bile acids and other metabolites in that area due to cholestasis. ^{29 30} Notably, this child has no current evidence for chronic liver or renal disease.

Fig. 2.

Light and electron microscopy imaging of respiratory and cholangiocyte cilia.

The treating physician suspected PCD and ordered bronchoscopy of the left lung with bronchus tissue biopsy for motility study and ultrastructure analysis. Nasal nitric oxide testing was deferred due to the difficulty of implementation on small children. It is important to note that nasal brushing is less invasive and typically preferred over bronchus biopsy; however, bronchial cilia evaluation holds a higher yield of adequacy as nasal cilia are more affected by chronic inflammation, trauma, and medications. The lung sample underwent two studies: high-speed cilia motility study (100–200 frames per second) with Nomarski polarizing optics, and ultrastructure analysis with an electron microscope (EM). Motility studies showed shortened cilia with restricted, asynchronous motility ( Videos 1 , 2 , 3 ; Fig. 2D left). Under EM, longitudinal profile of cilia appeared to be normal at their points of basal attachment ( Fig. 2D right). However, instead of normal respiratory cilia ultrastructure findings ( Fig. 2E ), this child's study revealed occasional well-oriented axonemes lacking entire outer arm or outer arm hooklets ( Fig. 2F ). These results support motile 9 + 2 cilia dysfunction in the lung.

Fig. 3.

Sanger sequence chromatograms of mutation sites for the patient.

The patient was subsequently referred to our care at Cincinnati Children's Medical Center, where he and his parents underwent a Heterotaxy, Situs Inversus and Kartagener's syndrome panel through PreventionGenetics. This comprehensive DNA testing consisted of next generation sequencing for 36 genes (e.g., DNAH5 , DNAI1 , FOXH1 , DNAAF1 , CCDC103 ; for a complete list, see Table 1 ) associated with PCD and/or laterality defects using exome capture probes, followed by Sanger sequencing (for uncaptured regions or regions with insufficient numbers of sequence reads) of pathogenic, likely pathogenic, and uncertain variants. Initial analysis of the DNAH5 gene indicated that the patient is heterozygous for c.8498G > A, a known pathogenic variant. Two additional variants were found in DNAH5 : c.1206T > A and c.7800T > G. Targeted Sanger sequencing for the three DNAH5 variants were performed on the parents to determine if the variants were found in cis- or trans-presentation in the proband. Sanger sequence chromatograms are reported in Fig. 3 for the patient and in Fig. 4 for the parents. The c.8498G > A variant was found to be inherited maternally. The c.1206T > A and c.7800T > G variants were both found to be inherited paternally. Thus, the known pathogenic variant was in trans presentation from two variants of uncertain significance (VUS), suggesting that one or both of the VUS are pathogenic. As both parents are asymptomatic carriers, the autosomal recessive transmission pattern of DNAH5 indicates that the patient is a compound heterozygote. The family's genetic panel indicated no other variants in DNAH5 or in the other 35 genes tested.

Table 1. Complete list of 36 genes assessed by the “Heterotaxy, Situs Inversus and Kartagener's syndrome panel” through PreventionGenetics.

Heterotaxy, Situs Inversus and Kartagener's syndrome panel (36 genes)
ACVR2B	AK7	ANKS6	ARMC4	CCDC103	CCDC114	CCDC151	CCDC39
CCDC40	CFAP298	CFAP53	DNAAF1	DNAAF2	DNAAF3	DNAAF4	DNAAF5
DNAH11	DNAH5	DNAI1	DNAI2	DNAL1	FOXH1	GAS8	GDF1
INVS	LEFTY2	LRRC6	MMP21	NKX2–5	NME8	NODAL	PIH1D3
SPAG1	TTC25	ZIC3	ZMYND10

Fig. 4.

Sanger sequence chromatograms of mutation sites for the parents.

Discussion

To our knowledge, this is the first time the pathogenicity of either c.1206T > A or c.7800T > G variants is reported in literature. Due to the rarity of PCD (0.01–0.02%) in the population and the expenses/challenges of genetic testing, we still do not fully understand the clinical importance of many VUS in associated genes such as DHAH5 and patients may not be able to receive a conclusive diagnosis of PCD or another ciliary disorder, even following genetic testing.

This is a unique opportunity to report a patient with compound heterozygosity in the DNAH5 gene, with a pathogenic c.8498G > A on the maternal allele and 2 VUS on the paternal allele. Suspicion of nodal 9 + 0 cilia defect was raised due to visceral heterotaxia, asplenia, and complex congenital heart disease. A diagnosis of PCD and motile 9 + 2 ciliopathy was supported by high-speed bronchus cilia motility studies, bronchial cilia ultrastructure, and genetic testing. We reasoned that, for the patient to exhibit the characteristics and complications of PCD, c.1206T > A (dbSNP: rs140782270) and/or c.7800T > G (dbSNP: rs369225476) could be pathogenic, one or both. While ClinVar-reported in-silico missense tests predict that c.1206T > A (p.Asn402Lys) has uncertain significance while c.7800T > G (p.Ile2600Met) is likely benign, these sources admit uncertainty. As such, we do not believe judgement can be presently made regarding which of these VUS, or if both, are pathogenic in causing PCD. With this paper, we hope to inform clinicians of these findings and acknowledge that additional discoveries of these VUS are required to establish pathogenicity. For instance, if another PCD patient is found to have one known pathogenic allele and c.7800T > G on the other, this would indicate that c.7800T > G is pathogenic. Likewise, if the symptomatic patient has one pathogenic allele and c.1206T > A on the other, this would suggest that c.1206T > A is the pathogenic variant. Lastly, if a symptomatic PCD patient is found with c.7800T > G on 1 allele and c.1206T > A on the other, this would suggest that both VUS are indeed pathogenic. Functional studies may also aid in proving variant pathogenicity; indeed, induced pluripotent stem cell (iPSC) model development with these DNAH5 VUS variants is currently underway at our institution to further characterize pathogenicity, along with observations on in-vitro development/function. iPSC modeling, along with CRISPR/Cas9, permits intentional, selective mutation of nucleotides, and visualization of resultant functionality. This method has precedent in other research areas and has been shown to be a promising VUS-risk assessment tool. ³¹

Conclusion

PCD is a rare, autosomal recessive disorder resulting from ciliary gene mutations, commonly in DNAH5 . Expanding our repertoire of variant locales and understanding their effects will improve the sensitivity of PCD diagnosis and treatment in the future. Our findings of c.1206T > A (p.Asn402Lys) and c.7800T > G (p.Ile2600Met) may also serve as a catalyst for discovery of additional pathological DNAH5 variants.

We also wish to remind clinicians that PCD belongs in the differential diagnosis of all infants with unabating cholestasis. Defects in nodal 9 + 0 cilia due to a variant, such as in DNAH5 , impair proper organ organization and may result in laterality defects and midline liver, which as noted above, are associated with biliary atresia. Defects in motile 9 + 2 cilia result in defective muco-ciliary escalators that increase risks of bacterial/viral infections. Both biliary atresia and infections are known etiology for bile flow obstruction and cholestasis. Though we suspect that this patient's DHAH5 variant, and the subsequent nodal 9 + 0 and motile 9 + 2 ciliopathy, is likely directly or indirectly complicit in this presentation, other etiologies (such as being small for his gestational age and amoxicillin usage) may contribute. It is also possible, though unlikely, that this patient has another gene mutation elsewhere specific to cholestasis that we have not assessed for variance. Notwithstanding, the discovery of this patient's refractory cholestasis prompted further investigation that revealed PCD mutations of clinical importance.