Primary+Ciliary+Dyskinsia

= **Primary Ciliary Dyskinesis** =

**Description**
Primary Ciliary Dyskinesia is broad genetic disorder that affects the ability of cilia to be motile. Consequently, it also affects a broad range of body functions including he aring, respiratory function, and reproduction. (Zariwala et al. 2011) Primary Ciliary Dyskinesia affects 1 in 20,000 people on average. (Meeks 2000) toc

**History of the Disease**
While the symptoms of PCD can be fairly distinguishing, because of the rarity of the disease, little historical literature exists. The disease was first described by Ukrainian physicist A.K. Zivert in 1904, but it was generally acknowledged when Manes Kartagener published a report detailing the pathology in 1933. (Kartagener 1933) This led to the term Kartagener’s Disease, which represents the classic PCD triad of situs inversus, chronic sinusitis, and bronchiectasis. Almost no history is known of Zivert (McManus 2004), and the disease itself has only recently gained interest because of genetic research.

**Signs and Symptoms**
PCD’s symptoms can include mucus buildup in the lungs, recurrent respiratory infections such as bronchitis, bronchiectasis, infertility in males, hearing loss, and irregularly beating or inefficient cilia. (Sommer 2010) A subset of PCD, Kartagener syndrome exhibits the same symptoms as above, with the addition of situs inversus creating a distinction between the two. (Unknown 2010, Olbrich 2002)

**Diagnosis, Treatment and Management**
Typically situs inversus is the easiest way to identify a PCD/Kartagener’s patient, though this must be in conjunction with the previous mentioned symptoms. Confirmation of a PCD diagnosis can be somewhat complicated, due to the ambiguous presentation of the disease and low incidence in populations. In one study, 32% of patients required 50 to 100 doctors visits before the disease was identified, with the a mean age of 10.9±14.4 years for diagnosis. (Somner 2011) One method is to perform a genetic screening to see if the patient has any recognized mutations for PCD. However, this is only partially effective as not all mutations have been identified. Other methods include immuno-fluoresence and transmission electron microscopy, as well as high speed video microscopy of the cilia’s beat frequency and pattern to check for abnormalities. (Armengot 2010) Lastly some studies have suggested that lowered nasal nitric oxide levels may indicate PCD. (Lundberg 1994) Treatment of PCD is not currently possible with existing technology. Currently, the only methods of avoiding PCD are through screening of potential candidates, and in-vitro fertilization. Management of PCD is difficult, but can be effective if PCD is caught early. While no treatment for the cause of the disease exists, it is possible to control it’s symptoms. Because of PCD’s affect on respiratory function and mucus removal, patients are recommended to follow procedures similar to those with cystic fibrosis. (Bouvagnet 2009) These include vigorous exercise, coughing, and breathing maneuvers designed to clear the lungs. Because of the high chance for bronchiectasis, patients must be prompt in their application of antibiotics if they exhibit signs of bacterial infections like bronchitis or otis media. (Benditt 2008) If the bronchiectasis has progressed too far it may be necessary in some cases to perform a lobectomy to prevent recurring infections. Those affected by PCD must also be careful to get routine immunizations against Pertussis, Haemophilus influenzae type b, as well as the Pneumococcal and annual influenza vaccines. Lastly, because of sinus and ear complications with otitis media, children with PCD may need hearing aids and speech therapy. (Zariwala 2011)

**Inheritance**
Primary Ciliary Dyskinesia is an autosomal recessive disease, comprised of 12 known genes. The disease exhibits the classic 25% normal, 50% carrier, 25% affected genotype that characterizes a autosomal recessive disease. (Zariwala 2011)


 * 1) Pedigree of inbred Israeli-Bedouin families with PCD. Stars represent assumed carriers, filled in squares or circles are affected,and dots represent carriers.

**Molecular Basis / Pathology of the Disease**
Primary Ciliary Dyskinesia is a mutation of any component of cilia that impairs it’s function. The human cilia is composed of 9 pairs of outer microtubules, and 2 inner microtubules, with the inner pair attached to the cilia’s shaft structure. The outer pairs generate shaft movement via several outer dynein arms that cause the pairs to contract vertically. This is combined with inner dynein arms that anchor the outer pairs to the inner microtubules, which then generate the beating movement. (Asai 2001) Since multiple proteins compose cilia, several genes can cause PCD to varying degrees. Of the related genes, DNAH5 and DNAI1 are the most prevalent at 15-21% and 2-9% of the total PCD affected population, respectively. (Zariwala 2011, Zietkiewicz 2010, Hornef 2006)   Other genes can include DNAH11, RSPH9, RPSH4A, or DNAL1, though their expression in the PCD population is less. (Mazor 2011, Castlemen 2009, Reish 2010)  Mutations to DNAI1 that caused PCD were two mis-sense variants, and of all the different mutations, most were found in intron 1 and exons 13, 16, and 17. (Zariwala 2006) DNAH5 mutations were primarily nonsense mutations, concentrated in exons 34, 50, 63, 76, and 77. (Hornef 2006) Both genes code for outer dynein arm proteins, meaning any mutation that disrupts the protein formation also drastically impairs cilia function. Specifically, DNAH5 codes for a large, heavy chain protein that link the microtubules to the central cilia pair. DNAL1, a different protein, links to the microtubules, and DNAH5, bridging the gap between the two as an intermediate chain. Other, lighter chain proteins also link DNAL1 and the microtubules. (Mazor 2011) Though there are several types of outer and inner dynein arms, any loss of function can cause a cilia to beat out of sync with it’s neighbors. Because cilia are only effective when working in concert, this can still allow the symptoms of PCD to arise. With oscillation impaired or stopped, mucus laden with bacteria that would otherwise be swept out of the respiratory system is allowed to build up. This lack of oscillation also causes auditory problems as cilia are vital to translating vibrations in the inner ear into nerve signals that allow hearing. With a single flagella powering sperm cells, damage to it’s ability to move can render males affected by PCD infertile. Lastly, cilia during embryogenesis determine the organization of body asymmetry, meaning damage to their movement can cause the classic Kartengers symptom of situs inversus to occur. Because PCD only encompasses genes related to sperm flagella and respiratory cilia, the disease pathology is well known, though the underlying genetic causes may be less so. 1. Molecular pathway for pathogenesis

**Recent Advancements in Research**
PCD research primarily focuses on identifying the genes involved with in the pathogenesis of the disease. Here, three articles will be examined to show the most recent work being done on PCD and it’s genetic components. **1. Deletions and Point Mutations of LRRC50 Cause Primary Ciliary Dyskinesia** **Due to Dynein Arm Defects** Genomic analyses were performed on a group of 58 participants exhibiting inner and outer dynein defects. The LRRC50 gene was examined because of it’s involvement with ciliary dysfunctions in zebra fish. Several patients with dynein arm defects were sampled, PCR was performed, with results indicating both point mutations and deletions were the cause of the interrupted LRRC50 These deletions also extended to a region on chromosome 16q24 associated with several types of cancer, suggesting that the 3 patients with these may be more at risk. 3 individuals also exhibited randomized body asymmetry, which is validated by the role of cilia in determining body asymmetry during development. This is demonstrated by the motile m onocilia orthologs in zebra fish being crucial in determining left right asymmetry. To confirm this, a 1260bp probe containing the 3’ UTR of Lrrc50 was hybridized with mouse embryos. As expected, the embryo showed expression at the ortholog of Lrcc50. Since PCD also involves respiratory cilia, expression of Lrrc50 in ciliated mouse respiratory cells was examined, and confirmed. To see the specific molecular affect of Lrrc50, immunofluorescence was performed on several dynein-arm proteins, with DNAH5 and DNAH9 heavy chains observed to be absent. DNALI1 was also seen to be absent, showing that Lrrc50 affected both inner and outer dynein arm chains. DNALI1, DNAH5 and DNAH9 were found in the cytoplasm instead, suggesting that Lrrc50 is involved in the preassembly of dynein arms. Because of the potential linkage to kidney disease seen in zebrafish models, patients kidney function was examined but no damage was exhibited, suggesting that Lrrc50 does not have a role in kidney function in humans. (Loges 2009) **2. DNAH5 Mutations Are a Common Cause of Primary Ciliary Dyskinesia with Outer Dynein Arm Defects** A PCD locus was localized on chromosome 5p18, and DNAH5 was identified as a potential candidate for examination. This was backed up by DNAH5 coding for a heavy chain of outer dynein arms (ODA), and 28% of 109 PCD affected families tested displaying DNAH5 mutations. Blood samples were taken from 134 PCD families, and 109 nuclear family members, with 47 families showing ODA defects. Genomic DNA was extracted from the samples, and then compared against 10 controls, and then used with reverse transcriptase to produce the cDNA necessary for analysis. Immunofluorescence and videomicroscopy were used to confirm the cilia were dysfunctional. Upon analysis of the cDNA, a heterozygous G>C mutation at codon 1730 was seen. Reverse PCR was performed, with two products - a 593 and 449bp fragment This mutation causes premature translation ending in the 449bp fragment because exon 13 is skipped when splicing. In several individuals with mutant DNAH5, analysis of respiratory cells showed that either DNAH5’s product accumulated in the cytoplasm or at the base of the cilia, but were not integrated into the ODAs. Of 9 individuals examined with inner dynein arm defects, none had DNAH5 mutations, suggesting that DNAH5 only affected ODA assembly. A population analysis was also undertaken, with 38 of 134 total families having DNAH5 mutations. Because DNAH5’s products were found, but not correctly localized, DNAH5 likely is involved in targeting microtubule centers along cilia. Mutant DNAH5 was also found to preserve the N-terminal region of the protein, indicating that the C-terminal is important in this localization. (Hornef 2006)
 * 1) The chromosome Lrrc50 is located on, with the specific region it inhabits expanded.
 * 2) 4 individuals with deletions of Lrrc50. Deletions are highlighted in gray.
 * 3) Genomic structure of HSDL1 and Lrrc50, with area of interest highlighted.

**3. Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1** Two consanguineous Bedouin families were identified with 3 members affected by PCD. Blood samples were taken, and genomic DNA isolated. Homozygosity for the mutation was assumed, with DNAH5 and DNAI1 being examined for linkage and found to be heterozygote. The DNA was analyzed for similarities between the patients, and a large homozygous portion of chromosome 14 on the rs17176306 and rs41471545 regions were found. Additional family members were tested, with a linkage found at the 14q24.2-q13.3 locus. Reverse transcription was then performed on the total RNA extracted, PCR was performed, and the products were sequenced. The mutation was found to be an A>G base change at codon 449. A protein model was constructed, and the conservation of the protein sequence was found to be highly conserved. The model also demonstrated that the swap of Asn for Ser caused by the mutation greatly affected the folding of the protein. Further analysis of the protein was done to see the effect of the mutation. PCR was performed, and RNA harvested from a healthy control and one of the patients. The product was then restricted, and tested for mutations introduced by PCR. HEK293T cells were transfected, and a seven-fold reduction in the quantity of mutated protein after 48 hours was observed compared to the wild-type protein. Testing was done with immunoblot dyeing, and the mutated protein was shown to be significantly more unstable after 3 and 6 hours, reducing in quantity by 40 and 94% at each measurement. To test the interaction of the dynein
 * 1) Immunofluoresence of DNAH5 (red), Axenomes (green), and Nuclei (blue). A is a healthy proband, with DNAH5 and the Axenomes properly attached to the cilia body in respiratory tract cells.
 * 2) Mutant DNAH5 is targeted to the cilia base, but accumulates there rather than moving to it’s proper location. DNAH5 is not attached to the ciliary axenomes at all.
 * 3) Patient contains a heterozygous mutation for DNAH5, explaining the attachment at both ends of the respiratory cell. Only partial amounts of DNAH5 contain the mutation, meaning the cell is still functional to some degree.

arms when combined with cilia, rat tracheas were prepared for use. The tracheas were used for axonemal extracts to be combined with immunoprecipitation of the mutated and healthy dynein heavy chains. When coimmunoprecipitated, DNAL1 had an 80% reduction in the amount of dynein heavy chain, and tubulin compared to the control. This suggests that the interaction between DNAL1, the tubulin, and the heavy chain is damaged. This however does not mean it is completely non-functional, which could indicate a semi-dominant genotype. This is not confirmed by the available data though, as the parents of the PCD patients did not exhibit any of the symptoms such as situs inversus or chronic respiratory disease. While their cilia were not examined, and thus this cannot be confirmed, it still remains unlikely. (Mazor 2011) **4. Other Research** While research on PCD is limited, and specific to the genes that interact with cilia, the subject of population penetrance is contested within the field. Because of the extreme rarity of patients, and the ambiguity of the symptoms, the actual number of PCD patients, and patients affected by a specific mutation, wildly fluctuate. Some studies deal with patients that may exhibit the founder effect, since they are primarily done with limited ethnicities such as Polish or Bedouin groups, given the rare nature of the disease. Zietkiewicz (2010) criticizes this, pointing out the anomalous variations of penetrance for different diseases in some populations such as the Swiss or Italians, but not others. He also challenges accusations of preselection, because by the nature of the disease researches are looking for those with ODA defects, which naturally limits the study sample. Recent research has also suggested that the penetrance estimation for certain genes may be askance, with Failly //et al.// (2008) showing that DNAI1 may be closer to 2% in the general PCD population. Other research has branched out beyond focussing on the protein components of cilia as the cause of PCD. Recent research by Milara (2010) has suggested a chemical pathway via adenylate kinase that regulates the transformation of 2ADP into ATP + AMP, which drives the movement of cilia. Down-regulation and up-regulation of the gene has shown that it is involved not only in the powering of the cilia, but also in the continued maintenance of the structure. While the linkage between the AK7 gene and PCD expression is not strong, it still shows that other pathways may explain the roughly 50% of PCD patients whose mutations have not been confirmed yet.
 * 1) Record of wildtype and mutant DNAL1 over the course of 6 hours. Mutant DNAL1 is shown to significantly degrade in comparison to it’s wild-type version.

**Population Genetics**
PCD population penetrance as a whole is hard to estimate, but Meeks (2000) places the number around 1 in 20,000, though that comes with the caveat that it is likely an underestimate. Bedouin populations have been noted as having unusual rates of PCD, with at least two studies examining PCD in their ethnicity. This is likely exacerbated by familial inbreeding practices because of their nomadic nature. Most PCD patients are predominately caucasian, with little variation between Poles, Germans, French, or British populations.

**Additional Resources**
[] **-** A website devoted to raising PCD awareness and helping families with PCD, as well as community support for those affected. [] - Basic overview of PCD symptoms and causes [] - General information on PCD from the NIH [|http://www.ncbi.nlm.nih.gov/books/NBK1122/#pcd.Molecular_Genetics] - NCBI page with greater detail on PCD.

**References**
Kartagener M (1933). "Zur Pathogenese der Bronchiektasien: Bronchiektasien bei Situs viscerum inversus". //Beiträge zur Klinik der Tuberkulose.// **83** (4): 489–501.

J. Ulrich Sommer, Kerstin Schäfer, Heymut Omran, Heike Olbrich, Julia Wallmeier, Andreas Blum, Karl Hörmann, Boris A. Stuck. 2010. ENT manifestations in patients with primary ciliary dyskinesia: prevalence and signifcance of otorhinolaryngologic co-morbidities. Eur Arch Otorhinolaryngol (2011) 268:383–388

J.O.N Lundberg, E. Weitzberg, S.L. Nordvall, R. Kuylenstierna, J.M. Lundberg, K. Alving. 1994. Primarily nasal origin of exhaled nitric oxide and absence in Kartagener’s syndrome. Eur Respir J, 1994, 7, 1501–1504

Orit Reish, Montgomery Slatkin, Daphne Chapman-Shimshoni, Arnon Elizur, Barry Chioza, Victoria Castleman and Hannah M. Mitchison. 2009. Founder Mutation(s) in the RSPH9 Gene Leading to Primary Ciliary Dyskinesia in Two Inbred Bedouin Families. Annals of Human Genetics (2010) 74,117–125

Javier Milara, Ph.D., Miguel Armengot, M.D., Ph.D., Manuel Mata, Ph.D., Esteban J. Morcillo, and Julio Cortijo, Ph.D. 2010. Role of adenylate kinase type 7 expression on cilia motility: Possible link in primary ciliary dyskinesia.Am J Rhinol Allergy 24, 181–185, 2010

Miguel Armengot, M.D., Javier Milara, Ph.D., Manuel Mata, Ph.D., Carmen Carda, M.D. and Julio Cortijo, Ph.D. 2010. Cilia motility and structure in primary and secondary ciliary dyskinesia.Am J Rhinol Allergy 24, 175–180, 2010

Nada Hornef, Heike Olbrich, Judit Horvath, Maimoona A. Zariwala, Manfred Fliegauf, Niki Tomas Loges, Johannes Wildhaber, Peadar G. Noone, Marcus Kennedy, Stylianos E. Antonarakis, Jean-Louis Blouin, Lucia Bartoloni, Thomas Nu ̈sslein, Peter Ahrens, Matthias Griese, Heiner Kuhl, Ralf Sudbrak, Michael R. Knowles, Richard Reinhardt, and Heymut Omran. 2006. DNAH5 Mutations Are a Common Cause of Primary Ciliary Dyskinesia with Outer Dynein Arm Defects. AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 174, 120-126, 2006.

Maimoona A. Zariwala, Margaret W. Leigh, Franck Ceppa, Marcus P. Kennedy, Peadar G. Noone, Johnny L. Carson, Milan J. Hazucha, Adriana Lori, Judit Horvath, Heike Olbrich, Niki T. Loges, Anne-Marie Bridoux, Gae ̈lle Pennarun, Be ́ne ́dicte Duriez, Estelle Escudier, Hannah M. Mitchison, Rahul Chodhari, Eddie M. K. Chung, Lucy C. Morgan, Robbert U. de Iongh, Jonathan Rutland, Ugo Pradal, Heymut Omran, Serge Amselem, and Michael R. Knowles. 2006. Mutations of DNAI1 in Primary Ciliary Dyskinesia. AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 174 859-866

Niki Tomas Loges, Heike Olbrich, Anita Becker-Heck, Karsten Häffner, Angelina Heer, Christina Reinhard, Miriam Schmidts, Andreas Kispert, Maimoona A. Zariwala, Margaret W. Leigh, Michael R. Knowles, Hanswalter Zentgraf, Horst Seithe, Gudrun Nürnberg, Peter Nurnberg, Richard Reinhardt, and Heymut Omran. 2009. Deletions and Point Mutations of LRRC50 Cause Primary Ciliary Dyskinesia Due to Dynein Arm Defects. The American Journal of Human Genetics 85, 883–889

Masha Mazor, Soliman Alkrinawi, Vered Chalifa-Caspi, Esther Manor, Val C. Sheffield, Micha Aviram, and Ruti Parvari. 2011. Primary Ciliary Dyskinesia Caused by Homozygous Mutation in DNAL1, Encoding Dynein Light Chain 1. The American Journal of Human Genetics 88, 599–607.

Mike Faillya Alexandra Saittaa Analia Muñoza Emilie Falconneta Colette Rossiera Francesca Santamariad Maria Margherita de Santie Romain Lazorc, Celia D. DeLozier-Blancheta Lucia Bartolonia Jean-Louis Blouina. 2008. DNAI1 Mutations Explain Only 2% of Primary Ciliary Dykinesia. Respiration 2008;76:198–204.

Ewa Ziętkiewicz, Barbara Nitk1, Katarzyna Voelkel, Urszula Skrzypczak, Zuzanna Bukowy, Ewa Rutkiewicz, Kinga Humińska, Hanna Przystałowska, Andrzej Pogorzelski, Michał Witt. 2010. Population specificity of the DNAI1 gene mutation spectrum in primary ciliary dyskinesia (PCD). Respiratory Research 2010, 11:174

Victoria H. Castleman, Leila Romio, Rahul Chodhari, Robert A. Hirst, Sandra C.P. de Castro, Keith A. Parker, Patricia Ybot-Gonzalez, Richard D. Emes, Stephen W. Wilson, Colin Wallis, Colin A. Johnson, Rene J. Herrera, Andrew Rutman, Mellisa Dixon,Amelia Shoemark, Andrew Bush, Claire Hogg, R. Mark Gardiner, Orit Reish, Nicholas D.E. Greene, Christopher O’Callaghan, Saul Purton, Eddie M.K. Chung, and Hannah M. Mitchison. 2009. Mutations in Radial Spoke Head Protein Genes RSPH9 and RSPH4A Cause Primary Ciliary Dyskinesia with Central-Microtubular-Pair Abnormalities. The American Journal of Human Genetics 84, 197–209

Heike Olbrich, Karsten Häffner, Andreas Kispert, Alexander Völkel, Andreas Volz, Gürsel Sasmaz, Richard Reinhardt, Steffen Hennig, Hans Lehrach, Nikolaus Konietzko, Maimoona Zariwal, Peadar G. Noone, Michael Knowles, Hannah M. 2002. Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left–right asymmetry. Nature Genetics, 30, 143-144 David J. Asai and Michael P. Koonce. 2001. The dynein heavy chain: structure, mechanics and evolution. TRENDS in Cell Biology Vol.11 No.5, 196-202.

Maimoona A Zariwala, PhD, FACMG, Michael R Knowles, MD, Margaret W Leigh, MD. Primary Ciliary Dyskinesia. 2011. [cited 2011, Dec 1.] Available from: [|http://www.ncbi.nlm.nih.gov/books/NBK1122/#pcd.Molecular_Genetics]

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