Variation in DNAH1 may contribute to primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD, CILD, MIM 244400) is a clinically and genetically heterogeneous disorder of motile cilia dysfunction typically caused by an autosomal recessive mode of inheritance. PCD is characterized by various clinical manifestations, including chronic sinusitis, otitis media and chronic bronchitis leading to permanent lung damage (bronchiectasis). Situs inversus occurs in approximately 50% of PCD cases, a cohort who are specifically sub-diagnosed with Kartagener Syndrome (KS) based on a triad of symptoms [1]. Clinically, PCD affected female patients can present with sub-fertility due to defective oviduct cilia and males can be infertile due to immotile sperm flagella [2]. Occasionally hydrocephalus arises from a decrease of cerebrospinal fluid flow as a consequence of ependymal cilia dysmotility [3,4]. Previous estimates of the worldwide incidence of PCD is 1:15,000-1:30,000 live births but these figures are thought to be underestimated due to under diagnosis [5] due to such reasons as unfamiliarity with the disease and non-distinct signs and symptoms [6]. However, this estimate is reported to be elevated in certain consanguineous and isolated populations [7,8]. Clinically, only half of PCD patients are diagnosed before 5 years of age and diagnosis is often delayed until adolescence, with approximately one-third of these patients at adulthood [6,9,10].

Motile cilia are organelles with a microtubule cytoskeleton core, known as the axenome [11] comprising of nine outer-doublet microtubules, which encircle a central microtubule pair (i.e., 9 + 2 pattern). Outer dynein arms (ODAs) and inner dynein arms (IDAs), which are attached to the peripheral microtubules, provide the power by hydrolyzing ATP to the cilia that generate the force for motility for a number of biological functions and modulate beat frequency [11]. Dynein arms contain varying numbers of heavy (responsible for motor activity), intermediate and light chains [12-15]. Radial spokes and central pair projections are also present in most motile cilia and are further structures known to regulate dynein function [16]. PCD is characterized by ultrastructural defects in the axonemal structure of the 9 + 2 pattern of motile cilia and sperm flagella, for example, a complete or partial loss of inner and/or outer dynein arms, central microtubular abnormalities and defects of radial spokes. Thus, such biological defects are demonstrated clinically by recurrent or chronic sinusitis and bronchiectasis caused by immotile cilia in the upper and lower airways and infertility due to impaired oviduct cilia and sperm flagella [16]. To date, mutations in 20 different genes involved in the structure and function of the motile cilia have been reported to cause PCD, including those that encode the dynein axonemal heavy chain (DNAH) proteins (CILD3 (MIM 608644), caused by mutations in the DNAH5 gene on chromosome 5p (one of the most common involved in PCD) and CILD7 (MIM 611884), caused by mutations in the DNAH11 gene on chromosome 7p21. Recently, Ben Khelifa and colleagues [17] reported that homozygous variants in DNAH1 in five unrelated males were the cause of infertility without any other PCD symptoms, a disorder they proposed to call “multiple morphological anomalies of the flagella (MMAF)”.

In this case report, using a combination of homozygosity mapping and whole exome sequencing methodology, we describe the identification of a novel variation (c.3460 A > C; p.Lys1154Gln) in the DNAH1 gene in PCD affected sisters born from a consanguineous union. In addition to the results described here, a previous study described that the phenotype of the DNAH1 mouse homolog MDHC7 knockout resulted in asthenozoospermia (decrease in the motility of the spermatozoa) and reduced tracheal ciliary beat frequency using a photo-electrical method [15] and by a study by Blouin and colleagues who identified a potential novel PCD locus on chromosome 3p21 [25] both indicative that aberrations in DNAH1 may cause PCD. Tissue expression studies showed that DNAH1 is heavily expressed in the testis and trachea, both of which contain axonemal structures that are altered in PCD patients [26]. The lysine residue altered in these patients is located in the highly conserved N-terminal stem of the DNAH1 protein. This component is known to interact with other heavy chains and additional essential subunits of the axonemal complex [27]. Hence, the substitution of a basic, strongly positive lysine to a polar uncharged glutamine residue may alter the 3D confirmation and stability of the protein structure; disrupt interaction with other dynein chains and arm subunits and in the function of this heavy chain with regards to ATP hydrolysis used in ciliary and flagellar motility. As mentioned previously [17], variants in DNAH1 have been reported to cause asthenozoospermia without any accompanying PCD symptoms. They suggested that DNAH1 function in ciliated cells is probably compensated by other HC dyneins, such as DNAH12 (MIM 603340) as it is the closest paralog. However, the authors noted that none of the parents of their subjects could be analyzed to confirm transmission and hence segregation of the identified variants nor could they exclude the possibility that some of the variants might be hemizygous with a deletion on the other allele. Furthermore, they could not obtain biological samples from their subjects to exclude a reduction of ciliary beats that was seen in the mouse knockout and proposed that future work would require them to perform thorough physical analysis of their male subjects to indicate whether their patients might be at risk of developing late onset PCD. It is pertinent to note that the patients in this study were female and all of the subjects in Ben Khalifa and colleagues were male, possibly suggesting for now that for unknown reasons, females may be more sensitive to a more severe phenotype and/or that the p.Lys1154Gln variant may have a more severe affect than the variants identified in the first study. In addition, the authors cannot exclude the probability that although this variant was the only one found and may play a role in PCD, it is possible that a pathogenic causal variant may be located in an uncharacterized gene or in a known gene that may have missed by the constraints of the methodology used in this study. Further investigation including functional analysis is necessary to determine the effect of this variant on the protein and to explore the presence of SNPs or molecular variants working in parallel with the p.Lys1154Gln variant causing the expanded phenotype seen in the patient described here. With regards to our proband, IVF was successful and pre-implantation genetic diagnosis was cancelled as genotyping of the husband indicated that he was wild-type normal (Figure 1C) and hence all offspring would be obligate carriers. The pregnancy continued uneventfully until term and the proband delivered a healthy baby boy by cesarean section.

In summary, we have identified PCD patients from the same family with a missense variation that segregates with the disease phenotype in the DNAH1 gene. In addition to adding DNAH1 as a gene that may be involved in giving rise to PCD, the clinical diagnosis and the subsequent genetic findings have translated into an overall positive and beneficial outcome for the index patient and family and will be of benefit for future preventative and counseling measures in the future.