The role of cilia in health and in primary ciliary dyskinesia
Cilia are highly conserved organelles playing important roles in the both human health and disease. Primary ciliary dyskinesia (PCD), also known as immotile cilia syndrome is an autosomal recessive condition which is characterised by abnormalities in the structure and function of cilia and flagella. The dysfunction in ciliary motility leads to a reduction in mucous clearance thus resulting in oto-sino-pulmonary infections whereas dysfunction in flagella leads to impaired sperm motility thus causing infertility in male sufferers. 50% of PCD patients also have situs inversus totalis in which all the visceral organs are on the opposite side. This combination of conditions is called Kartagener syndrome.
In this structured review, I aim to emphasise the clinical implications of mutations and the resultant ciliary defects with regards to PCD. Research in this area is of great importance as PCD is a multisystem affecting disease that causes a wide range of symptoms putting the patient at risk of more serious complications. In order to understand the patterns of symptoms that occur in PCD, I will begin by explaining the normal biological construction and mechanism of action of cilia. I also aim to collate the most relevant and recent data regarding the diagnosis and management of the disease. Finally, future direction with regards to the detection and management of this condition will be discussed and key areas that need further investigation highlighted.
Structure and function of cilium
Cilia and flagella are structurally similar and the terms are often used interchangeably. Cilia are small organelles that project from the cell surface and the structure and proteomic composition have been conserved greatly through evolution from unicellular organisms (e.g. Chlamydomonas reinhardtii) to the cilia we see in humans today. This suggests that study of proteins in lower eukaryotic organisms can be used to gain an insight into the human orthologous proteins.
Cilia are made up of one of two characteristic arrangements of microtubules depending on their function. These are called the 9 + 0 (sensory cilia) and 9 + 2 (motile cilia) arrangements both of which contain nine microtubule doublets around the periphery and only differ from one another due to either the absence or presence of a central pair of microtubule singlet respectively (1, 2). The doublets are called microtubule A and B and connect with neighbouring doublets via nexin links which limit the amount they move.
Figure 1. Schematic representation of the ultrastructure of a ciliary axoneme. It illustrates the arrangement of the peripheral nine microtubule doublets (A & B) surrounding a central pair of singlet microtubules interconnected via dynein arms, nexin links and radial spokes.
Primarily, the intraciliary structures responsible for motility are the dynein arms, which are present in the 9 + 2 arrangement and subdivided into inner (IDAs) and outer (ODAs) dynein arms. These aid in moving the microtubule doublets relative to one another (3). The dynein arms are attached to microtubule A of the doublet and are composed of different sized polypeptides (heavy, intermediate and light). Due to the similarities in ciliary composition in Chlamydomonas and humans, most of our knowledge we now possess about cilia originates from studies of this unicellular organism. These studies began to identify identical or similar proteins to those that had been found in Chlamydomonas possessing the knowledge about their ODA structure consisting of three heavy chains (a-, b- and g-HCs), two intermediate (ICs) and nine light chains (LCs) (4). This study revealed that humans possess five orthologs of the b- (DNAH11, DNAH17, DNAH9) and g-HCs (DNAH5 and DNAH8) (4). Each of these orthologs appears to have specific motility functions dependent on where the cilia are located in the body. Knowledge about the presence and location of these is important in enabling one to deduce its function as a direct result of any clinical manifestation that occurs from its absence. IDAs on the other hand appear to exist as one of seven isoforms thus making them much more complex both structurally and functionally in comparison to ODAs. As depicted in figure 2, one of these isoforms is two-headed (I1) and six are single-headed. IDAs are predominantly responsible for the bending pattern of cilia whereas the frequency and force with which the cilia beat is controlled by ODAs.
These dynein arms are generally absent in cilia with the 9 + 0 arrangement, hence making them nonmotile. However, nodal cilia in the developing embryo are an exception to this as they have the 9 + 0 arrangement but they do also possess dynein arms, which make them motile thus allowing them to instigate fluid flow in the node as will be discussed in more detail later (5, 6). Some of the studies linking Chlamydomonas and human cilia will also be discussed later in this review with regards to defects that are seen within dynein arms.
Figure 2. Schematic representation of ODA and IDA structure. The ODA of Chlamydomonas (green) is composed of three HC (a, b and g), two IC and nine LC. However, IDAs are known to be in one of seven isoforms, one double-headed (light orange) whilst the other six are single-headed (dark orange). The numbers shown within the oval shapes are the molecular weights of the known components.
The mechanism by which the dynein arms move is similar to that of the actin-myosin complex. This is because attachment of ATP to the dynein arms allows the free end of the dynein to connect to microtubule B on its neighbouring doublet, thereby initiating movement. The radial spokes interconnect the central pair to the outer microtubules and is responsible for controlling the movements. The inner structure of the cilia explained thus far is enclosed within a ciliary membrane that controls the sensory functions as it contains receptors and channels that help with transduction of signals (7, 8). The cilia are anchored in position by the basal body.
There are many motile cilia per cell in comparison to sensory cilia which are usually one per cell. The cilia are ideally located depending on their function. For example, motile cilia are found on the surface of the epithelial cells lining the respiratory tract, in the fallopian tubes and on ependymal cells in the ventricles of the brain whereas the sensory cilia are positioned on the principal cells of the kidneys and ganglion cells in the eye.
Cilia are present on almost every cell in the body and play an important role in health. If they become dysfunctional, they can result in a variety of syndromes as highlighted in the table below (9). As the diverse range of ciliopathies is far beyond the scope of this review, I will be concentrating on PCD.
Primary Ciliary Dyskinesia
Primary ciliary dyskinesia (PCD) is a multi organ affecting disease predominantly inherited in an autosomal recessive pattern (10-12). However, it has been shown in rare cases to be inherited in an autosomal dominant or X-linked pattern too (13). The prevalence figures for PCD vary between populations ranging from 1:15000 to 1:30000 but there seems to be a higher incidence within populations in which a high proportion marry consanguineously (14).
PCD is a multi-complex condition presenting in a number of different ways. To truly comprehend the complicated phenotype, it is important to understand normal ciliary structure and function. This knowledge can then be used to deduce the reasoning for each of the phenotypes.
One of the most common symptoms includes recurrent oto-sino-pulmonary infections due to dysfunctional motile cilia, which no longer beat in a coordinated to augment airway clearance. The pulmonary infections eventually lead to bronchiectasis, which is localised permanent dilatation of the bronchi, usually occurring secondary to a bacterial infection (11, 15).
During normal body development, certain organs in the body exhibit asymmetry. This is due to a structure present in the embryo called the embryonic node which also possesses cilia. Nodal monocilia are normally responsible for the clockwise motion and leftward flow of embryonic fluid, also known as nodal flow. This is essential in initiating the correct localisation of visceral organs and the heart as depicted in figure 2A. Therefore, if these cilia are unable to carry out their motile function, the flow is disrupted and depending on the direction of flow which is determined randomly, it will result in situs solitus (normal localisation of organs) or situs inversus (mirror image) (15, 16) When PCD and situs inversus coexist in a patient, they are said to have Kartagener's syndrome (17). Half of the PCD population have situs inversus.
Figure 3. Role of nodal monocilia in determining left-right body asymmetry. (A) A mouse embryo showing location of nodal monocilia and how their movement causes leftward flow of embryonic fluid, as indicated by the red arrows. (B) It represents the randomised left-right positioning of organs within the body if nodal flow is impaired, hence either resulting in situs solitus or situs inversus.
Cilia/flagella also have a role to play in reproductive organs with regards to motility. Any abnormality affecting its ability to function in this role will result in a reduction in fertility. Males affected by PCD tend to be infertile due to immotility of sperm whereas affected females have lower fertility due to cilia not moving the oocytes effectively along the Fallopian tubes (18, 19).
Another clinical manifestation that has been associated with PCD in rare instances is hydrocephalus (20-23). This implies that cilia lining the ependymal cells in the brain are not necessarily always affected if the respiratory cilia are immotile (15). However, if they do become dysfunctional, they will reduce flow of cerebrospinal fluid within the ventricles of the brain.
Many ultrastructural defects have been associated with PCD. Abnormalities evident in human PCD subjects include dynein arm defects either individually or in both ODA and IDA, and defects in radial spokes, nexin links and central microtubule pairs. The most prevalent defects, up to 80% involve the dynein arms with ODAs being most affected (24, 25). The resultant cilia are immotile (25-27). However, if IDAs are the ones possessing the defect, then the amplitude of each beat will be reduced as shown by Chilvers et al. (26) and in comparison if the central pair of microtubules is affected, then the ciliary beat pattern will be altered from its norm (24).
These abnormalities in the structure of cilia are predominantly caused by specific genes which will be discussed later. In some PCD cases however, ultrastructural defects are not evident. This implies that defects are still present but are having a profound effect on the functionality of the cilia in these cases rather than on its structural composition.
Identification of loci and structure of genes
Homozygosity mapping studies and the candidate gene approach are useful methods for obtaining information regarding loci and structure of genes involved in PCD. In mapping studies, a large consanguineous family with PCD is identified. Homozygous regions of the chromosomes in all of the affected patients are located, thus enabling the gene responsible for the defect to be "mapped" (28, 29). The candidate gene approach on the other hand is a viable method because of the close relationship between the structure and function of cilia in Chlamydomonas models and humans. This approach makes use of the knowledge we possess about certain genes being responsible for specific defects in Chlamydomonas. The human genes responsible for causing equivalent defects are then potential candidates for PCD.
3 genes in particular have been shown to be present with mutations in PCD and these are DNAI1, DNAH5 and DNAH11 (3, 30). These will be discussed further in the following sections.
Genes implicated in PCD
DNAI1 was the first human gene that was discovered to be harbouring mutations associated with PCD in which ultrastructural defects were also present (31). It is the human ortholog of the Chlamydomonas ODA protein IC78 and has been located at the chromosomal region 9p13-p21. It comprises of 20 exons and encodes for a protein called dynein intermediate chain 1, which like its ortholog in Chlamydomonas is found in ODAs. Hence, mutations involving this gene have shown associations with ODA defects (32). DNAI1 mutations are the initiators that result in an individual having either situs solitus or situs inversus (30).
DHAH5 is the human protein coding ortholog of the Chlamydomonas g-HC and is located on chromosome 5 in a specific region called 5p15 (28). This gene was discovered following a mapping study on a large consanguineous PCD family who had either complete or partial absence of ODAs (28, 33). DNAH5 consists of 80 exons and spans 250kb (34). It has been shown that the ultrastructural phenotype bears some correlation to the genotype. For example, the complete or partial absence of ODA in patients carrying mutations in DNAH5 resulted from either premature translation termination or splice site mutations respectively (35). With splice site mutations it is predicted that an exon is lost which results in a shorter than normal ODA being produced.
DNAH11 is a gene that encodes for heavy chain 11 and comprises of 82 exons spanning more than 353kb of chromosome 7. It is localised in the region 7p21. The first patient that possessed a mutation in DNAH11 had a phenotype that consisted of situs inversus, cystic fibrosis and paternal uniparental isodisomy, which is when both chromosomes in a pair are inherited from the father. As situs inversus is seen in patients with DNAH11 mutations, it implies that this gene may have a link to the functioning of nodal monocilia. This is because these structures play a vital role in determining left-right body asymmetry. In heterozygous DNAH11 mutations there are no associations with the dynein arms and the remainder of the axonemal ultrastructure also appears to be absent of any defects. Therefore, the cilia still have motility but there beating pattern differs with a decrease in their ability to bend and a faster than normal beat is also seen (36). The faster beat may be a compensatory mechanism to overcome the reduction in bending ability to help maintain enough ciliary motility to function normally.
Other genes that have also been associated with PCD include RPGR, TXNDC3 and DNAI2 (37). A variety of other candidates genes are under study to see if they are implicated in PCD sufferers, but as yet no associations have been derived.
Due to the complexity of the disease, a number of diagnostic options are available for use and the main ones will be discussed in this section. As mentioned earlier, PCD has a characteristic presentation, which includes oto-sino-pulmonary infections, situs inversus, infertility and hydrocephalus. If these symptoms are present in a patient, it helps make diagnosis simpler. It is crucial that an early diagnosis in all patients, especially children is made to prevent irreversible damage from occurring such as bronchiectasis.
Transmission electron microscopy (TEM) is the standard method used to localise any ciliary ultrastructural defects seen in PCD patients. I believe as with any method, TEM has its limitations. These include the requirement of specialist trained personnel throughout the process of obtaining the specimen (tissue) and images as well as analysing them correctly. Also, TEM is not perfect in that it cannot detect all defects at the ultrastructural level such as those present in IDAs. TEM is also made redundant in patients who as discussed previously in this review have no ultrastructural defects.
High speed videomicroscopy is a technique that analyses the beat pattern of cilia, which also as shown earlier can be predicted from the type of ultrastructural defect present. Abnormal ciliary beating patterns that are visualised range from a circular motion to total immotility. However, it is important to know that normal ciliary beating can occur despite the presence of defects (38).
Another characteristic finding in PCD patients includes low levels of NO (nitric oxide) (38-42). Normally, NO has been shown to upregulate and increase ciliary beat frequency (3). Therefore, decreased ciliary motility seen in PCD patients may be associated with the low NO levels. Although the mechanism by which it arises is not yet fully understood, it is a vital diagnostic tool.
Finally, immunofluorescence imaging has also been used on respiratory epithelial cells to localise intraciliary components such as the ODA heavy chains, DNAH5 and DNAH9 through the use of specific antibodies (43). This method has many advantages including the non-invasive means of obtaining the necessary cells via nasal brushings and the ability to detect changes along the whole of the axonemal length. I believe as the number of antibodies being used for this procedure increases in the future, the number of defects detectable using this technique will also increase.
PCD patients should be under review of a multidisciplinary team. The team members should include chest physicians, physiotherapists and PCD specialists. The patient should also have access to fertility clinics and counselling services in the community. The primary aim of treatment with regards to PCD is to aid in the prevention of permanent lung disease as well as other preventable complications.
When PCD patients present with deterioration in lung function, aggressive antibiotic treatment should be administered. This should be coupled with physiotherapy to promote airway clearance. Many PCD sufferers will need support and information regarding infertility and assisted conception techniques.
Although it may not seem like it forms a direct part of the clinical management plan, it is essential that psychosocial aspects of the patients care are not neglected. Information regarding application for benefits as well as counselling services in order to help the patient cope with their condition could prove beneficial. Another crucial aspect of care is educating people with whom the patient will have large amounts of contact, such as school teachers, about any potential problems that may be faced.
It is essential to maintain regular contact with PCD patients in the form of follow-up appointments to ensure good health as both early diagnosis and effective management improve prognosis (44).
The management discussed thus far is emphasising how to deal with patients presenting with PCD and common symptoms associated with it but no method has yet evolved to correct the fundamental errors that cause the ciliary dysfunction. Gene therapy has been a method used in cystic fibrosis but with no success as useful protein expression could not be established. However, pharmacological means of restoring expression of functional proteins is now under way to determine its use in this situation.
Animal models and their use in PCD
Animal model systems play a crucial part in obtaining information that can be extrapolated and applied to humans. Chlamydomonas has been one of the main models used for cilia. This is due to the high levels of evolutionary conservation of both ciliary structure and function from these organelles to humans. Chlamydomonas is a good model system with respect to obtaining information about the biochemistry of cilia but applying it directly to a complex multicellular organism for me seems to have its limitations and thus should be done with great caution.
For example, although a high level of conservation is clearly evident, humans could have developed human-specific proteomic changes over time that may be overlooked or not even discovered if too much emphasis is placed on similarities and not enough on differences between Chlamydomonas and humans. Also, ciliary dysfunction in a unicellular organism is not likely to cause complex phenotypes as is present in human PCD patients. This is because there are a large amount of cilia carrying out a diverse range of functions in multicellular organisms like human beings. This is even more of a reason to ensure that assumptions are not made without fully researching and obtaining reliable information regarding true correlations between humans and lower eukaryotes.
Mouse models have been used to demonstrate PCD in a multicellular organism other than humans (45). A disadvantage with this is that knockout mice have to be created which makes it a more expensive and complex model to use. However, as mice are evolutionarily closer to humans, these models can assist in understanding the multi-complex phenotypes seen in humans. Other PCD animal models that have also been used include rats, dogs and pigs which will not be discussed further in this review but can be found from the relevant references mentioned here (46-48).
Conclusions and future directions
Our knowledge of PCD has greatly increased over the past decade with the genotype-phenotype correlation being better understood and more information about diagnostic and therapeutic options becoming available.
In the future, the key things to look out for are the pharmacological developments that have the potential to assist in protein expression, which would help overcome ciliary dysfunction. However, they will need to undergo extensive trials to ensure their safety for use. I strongly believe if this method is successful in achieving the intended goal, these drugs will have the potential to act as curative agents in PCD patients. Another key issue that is under rigorous study at the moment is the identification and confirmation of more PCD causing genes. This knowledge is crucial to allow for the development of suitable and effective screening methods and therapeutic agents.
Finally, the initial aims I set out to achieve have been completed to the best of my ability through the use of multiple sources. To summarise, this review was needed to understand the literature and to draw upon evidence to highlight the importance of ciliary structure and function in health, defects seen in PCD and the genotype-phenotype mechanisms involved. I believe that there is enough evidence to show this at the level required for this review but to fully understand the molecular basis of PCD, more research is required.
I thank Dr Paul Mckean for all his guidance and support throughout this review. His input has been invaluable and much appreciated.
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