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Original Research: Pediatrics |

Bardet Biedl SyndromeBardet Biedl Syndrome: Motile Ciliary Phenotype: Motile Ciliary Phenotype FREE TO VIEW

Amelia Shoemark, PhD; Mellisa Dixon, PhD; Philip L. Beales, MD; Claire L. Hogg, MBChB
Author and Funding Information

From the PCD Diagnostic Team (Drs Shoemark, Dixon, and Hogg), Royal Brompton and Harefield NHS Trust; the National Heart and Lung Institute (Dr Shoemark), Imperial College; the Institute of Child Health (Dr Beales), University College London; and Great Ormond Street Hospital NHS Foundation Trust (Dr Beales), London, England.

CORRESPONDENCE TO: Amelia Shoemark, PhD, Electron Microscopy Unit, Royal Brompton Hospital, Sydney St, London, SW3 6NP, England; e-mail: a.shoemark@rbht.nhs.uk


FUNDING/SUPPORT: The authors have reported to CHEST that no funding was received for this study.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2015;147(3):764-770. doi:10.1378/chest.13-2913
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Published online

BACKGROUND:  Cilia line the surface of the respiratory tract and beat in a coordinated wave to protect the lungs against infection. Bardet Biedl Syndrome (BBS) is a rare condition attributed to cilia dysfunction. Murine models of BBS suggest a respiratory phenotype; however, no reports have studied the translation of these findings in patients.

METHODS:  We assessed the clinical symptoms of motile cilia dysfunction and the histology of ciliated respiratory epithelium in patients with BBS.

RESULTS:  We report an increased prevalence of neonatal respiratory distress at birth (12%), general practitioner-diagnosed asthma (21%), otitis media (33%), and rhinitis (36%) in patients with BBS. These symptoms, however, occurred at a significantly reduced prevalence compared with patients with known motile cilia dysfunction (primary ciliary dyskinesia). Respiratory epithelial assessment revealed cellular damage, significant ciliary depletion (on 60% of ciliated cells), and goblet cell hyperplasia in patients with BBS (50% goblet cells). These findings were quantifiably similar to those of patients with asthma (P > .05). Surprisingly, motile cilia function and ultrastructure were grossly normal with the exception of occasional unique inclusions within the ciliary membrane.

CONCLUSIONS:  In conclusion, motile ciliary structure and function are essentially normal in patients with BBS.

Figures in this Article

Bardet Biedl Syndrome (BBS) is clinically characterized by rod-cone dystrophy, truncal obesity, postaxial polydactyly, cognitive impairment, genital anomalies, and renal abnormalities.1 The phenotype of this genetic condition is thought to occur because of a loss of function in nonmotile primary cilia resulting from an absence of BBS proteins. These proteins are located at the base of the cilium, some in a complex called the BBSome, which is involved in the movement of particles in and out of the cilium by a process known as intraflagellar transport.1 Dysfunction of nonmotile primary cilia in this condition is well described; however, evidence from murine models suggest that BBS proteins are also colocalized with motile cilia on the epithelial surface of the respiratory tract, suggesting that this ciliopathy may also affect motile cilia.2 Murine models with defects in BBS proteins 1, 2, 4, and 6 demonstrate dysfunction of the ependymal cilia of the brain, sperm flagella, and motile cilia in the respiratory tract.2,3 Reports show slow ciliary beat frequency within the respiratory tract increased variation in ciliary length and swollen, paddle-shaped cilia tips containing vesicles on electron microscopy.2 The translation and clinical relevance of these murine findings have never been reported in human patients with BBS.

Dysfunction of motile cilia in humans usually results in a well-defined phenotype known as primary ciliary dyskinesia (PCD). Symptoms include neonatal respiratory distress, otitis media, rhinosinusitis, and chronic wet cough. Recurrent chest infections eventually lead to permanent scarring of the lung in the form of bronchiectasis. Motile cilia structure is similar to the flagella of sperm tails, and consequently, men with PCD are often subfertile.4 Occasionally, PCD has been reported in patients with nonmotile cilia dysfunction.5,6 The aim of this study was to assess the structure and function of respiratory ciliated epithelium and the clinical implications of respiratory ciliary dysfunction in a cohort of patients with BBS.

Subjects

A retrospective review of clinical data was analyzed for all patients attending BBS clinics at Great Ormond Street and Guys Hospital who were referred for respiratory cilia function tests. Results from 46 patients were analyzed, of which 24 were men (57%). Ages ranged from < 1 year to 48 years, with an average age of 22 years.

Study Design

Respiratory history was recorded from patients with BBS and their families, concentrating on factors implicated in motile respiratory ciliary dysfunction such as respiratory distress in the neonatal period; frequency of chest infections; and ear, nose, and throat symptoms. Patients were screened for ciliary dysfunction using a nasal nitric oxide (NO) screening test. Individuals in which PCD was indicated because of symptoms leading to high clinical suspicion or low nasal NO (< 250 parts per billion [ppb]) were followed up with a nasal brush biopsy (n = 16). Microscopy assessments of respiratory cilia were made by a blinded observer and were compared with three groups: patients with PCD and static cilia caused by an outer dynein arm defect, patients with asthma, and healthy control subjects.

Investigations

Clinical tests for ciliary dysfunction were performed according to national and international standards for the diagnosis of PCD following the standard operating protocols of the Royal Brompton Hospital, a National Health Service nationally designated specialist diagnostic center.7 These are described in the following sections.

Nasal NO:

Nasal NO was measured by chemiluminescence on a portable Logan analyzer at a flow rate of 250 mL/s (Logan Research Ltd). Patients were asked to hold their breath, and NO was measured when there was a 10-s plateau in value. Correct technique was assessed by a simultaneous CO2 trace. When subjects were unable to perform breath hold with satisfactory technique (n = 7), a tidal breathing method was used, in which subjects breathed normally with their mouths open. NO testing was not performed in children under 2 years of age.

Nasal Brush Biopsy:

Nasal brush biopsies were collected from the nasal inferior turbinate using a modified 3-mm bronchial cytology brush (Diagmed Healthcare) and were suspended in Media199.

Light Microscopy:

Nasal brushings were assessed for ciliary beat pattern and frequency using high-speed video microscopy.8 Cilia were recorded at 37°C under a 100 × oil immersion lens using a high-speed video camera at 500 fps (Fastcam Troubleshooter XS; Lake Imaging Systems). Movies were then played back at 60 fps, and motion analysis was performed on the top and side profiles.

Electron Microscopy:

Nasal brush biopsies were fixed in 2.5% glutaraldehyde in cacodylate buffer and were processed as previously described.9 Briefly, cells were washed in sodium cacodylate buffer, postfixed with 1% osmium tetroxide, and centrifuged in 2% agar to generate a pellet. Using a series of increasing concentrations of methanol followed by propylene oxide, cells were dehydrated before being embedded in Araldite resin. Sections of 70 to 90 nm in size were cut using a Reichert Ultracut-E ultramicrotome, mounted onto copper grids, and stained with methanolic uranyl acetate and lead citrate. Assessment of the respiratory epithelium and ciliary ultrastructure was made on a Hitachi 7000 transmission electron microscope. A clinical electron microscopist blinded to the case information quantified cells, determined their microtubular arrangement in the axoneme, and investigated the presence of dynein arms.

Ethics Statement

This study was conducted in accordance with the amended Declaration of Helsinki. The National Research Ethics Service Committee London, Bloomsbury, approved the protocol (study number 08/H0713/82). Signed patient consent was not required.

Patients With BBS Report Respiratory Symptoms

Patients with BBS report an increased prevalence of neonatal respiratory distress at birth, general practitioner (GP)-diagnosed asthma, and rhinitis compared with the general population. These symptoms, however, occurred at a significantly reduced incidence compared with patients with known motile cilia dysfunction (PCD) (Table 1).1016

Table Graphic Jump Location
TABLE 1 ]  Prevalence of Symptoms Associated With Motile Ciliary Dysfunction in Patients With BBS Compared With the General UK Population and Patients With PCD1016

Data are presented as % unless indicated otherwise. BBS = Bardet Biedl Syndrome; ENT = ear, nose, and throat; GP = general practitioner; NA = not available; PCD = primary ciliary dyskinesia.

There was no increase in the reported frequency of chest infections or cough, and cough was not wet or productive. Rhinitis was reported to be seasonal in five patients and associated with sinusitis in two patients.

Otitis media sufficient to require tympanostomy tube insertion was also more prevalent in patients with BBS than in the general UK population, but was less prevalent than in patients with PCD. In the BBS cohort, 11 tympanostomy tube insertions were considered successful, one had to be repeated, and in two, the outcome was unknown at the time of the study. This is in contrast to PCD, in which persistent perforation of the eardrum is not uncommon after surgery. No patients with BBS had hydrocephalus.

Nasal Respiratory Epithelium Demonstrates Ciliary Depletion and Goblet Cell Hyperplasia

Epithelial strips obtained from patients with BBS demonstrated an increase in the number of mucus cells and a decreased number of ciliated cells compared with the healthy control group and the PCD group. Quantifiably, the changes seen in BBS were similar to those in patients with asthma. These data are shown in Figure 1.

Figure Jump LinkFigure 1 –  Epithelial cell differentiation. A and B, Patients with BBS and asthma had fewer ciliated epithelial cells and more mucus cells compared with healthy control subjects and patients with PCD. D-F, Low-magnification electron micrographs of nasal epithelial strips from patients with BBS, demonstrating (D) intercellular spaces and (D and E) blebbing and goblet cell hyperplasia. C and F, When ciliated cells were present, they were often depleted of cilia. Short cilia in F probably represent regeneration. G and H, Healthy, well-ciliated epithelial strips for comparison. Scale bar = 10 μm. BBS = Bardet Biedl syndrome; PCD = primary ciliary dyskinesia.Grahic Jump Location

Epithelial strips typically exhibited signs of cellular damage characterized by intracellular spaces and epithelial blebbing. On the small number of ciliated cells that were present, the number of cilia was also often depleted. Despite ciliary depletion, signs of ciliogenesis, in the form of granular bodies, shortened cilia, and subepithelial ciliary components, were present, suggesting an active ciliary regenerative process.

Motile Respiratory Cilia Function Is Unaffected in BBS
Nasal NO Screening Test:

Nasal NO was within the normal range (400-1,000 ppb) in the majority of individuals with BBS. In six subjects, nasal NO was < 250 ppb. Five of these patients had nasal symptoms.

Ciliary Beat Frequency and Length:

Ciliary beat frequency and length were assessed by light microscopy. There was no significant difference in the mean values of ciliary beat frequency (9.5 Hz) or ciliary length (6.5 μm) from patients with BBS compared with control subjects and patients with asthma (e-Table 1).

Ciliary Beat Pattern:

The ciliary beat pattern was mostly coordinated and effective. Occasional nasal epithelial strips contained static cilia, short cilia, or dyskinetic ciliary beat. These changes are consistent with changes seen in patients with chronic respiratory conditions or upper respiratory tract infection (Videos 1-3).

Video 1.

Normal Ciliary Beat in Healthy Control

Video 2.

Normal Ciliary Beat in BBS

Video 3.

Ciliary Depletion and Short Cilia in BBS

Motile Cilia Axonemal Ultrastructure Is Unaffected in BBS

Electron microscopy revealed normal 9 + 2 axonemal structure in the majority of cilia cross sections (Fig 2). Inner and outer dynein arms were present on all cross sections. No abnormality of structure or orientation of the basal body or basal foot structure was observed. Quantification of axonemal features (e-Table 2) showed that all parameters were within the normal range, with the exception of occasional unique inclusions.

Figure Jump LinkFigure 2 –  Inclusions within the ciliary membrane in respiratory motile cilia from patients with BBS. A, At low magnification, inclusions are indicated by arrows. A insert, D, and E, At higher magnification, it can be seen in cross section that inclusions vary in their number, size, and electron density. F, These structures were also seen in longitudinal section within the cilium. B, Visually similar structures were observed within the cytoplasm of ciliated epithelial cells at low magnification indicated by black arrows and at higher magnification (B insert). The white arrow indicates granular bodies associated with ciliary regeneration, frequently seen in these samples. C, Axonemal structure appeared to be normal in patients with BBS. In this example, both dynein arms, radial spokes, and nexin dynein regulatory complex are present. Scale bars represent 500 nm in low-magnification images (A and B) and 100 nm in images C and F. See Figure 1 legend for expansion of abbreviation.Grahic Jump Location
Unique Ultrastructural Inclusions Are Found Within the Ciliary Membrane in BBS

The inclusions found within the ciliary membrane varied in their electron density, size (30-200 nm), and number (1-50 per cilium) (Fig 2). These structures were seen in 50% of BBS cases. They were found in < 1% of cross sections. There was no relationship between the presence or absence of inclusions and the age, sex, NO result, or ciliary beat frequency of the patient. There was a relationship between the amount of material available for assessment and the presence of these features. All samples in which inclusions were absent were from grids with < 100 cross sections available for assessment.

In an observer-blinded comparison, no similar structures were found within the ciliary axoneme in 10 normal control subjects, 10 patients with asthma, 10 patients with PCD, 10 patients with chronic respiratory infections, or eight samples from respiratory patients couriered from Great Ormond Street Hospital to Royal Brompton Hospital for possible PCD diagnosis (to exclude possible acquired defects during transport). Similar vesicular structures were seen within the cytoplasm of ciliated cells from patients in all groups.

In summary, patients with BBS have a high prevalence of asthma, otitis media, and rhinitis. This is reflected in the nasal epithelial histology, which shows damaged epithelium and goblet cell hyperplasia. Respiratory motile cilia function and structure are largely normal in patients with BBS; however, a small proportion of cilia contain inclusions within the ciliary membrane, unique to BBS and visible by electron microscopy.

BBS proteins form a structure known as the BBSome, located at the base of the cilium; it controls the movement of particles in and out of the cilium by intraflagellar transport. We speculate that the inclusions seen in the motile cilia of patients with BBS may be vesicles that arise because of poor control of the trafficking of proteins in and out of the cilium. Further investigation into the nature of these inclusions may reveal clues into the function of BBS proteins in motile cilia. Similar structures have been reported in murine models of BBS, and the similarity of these findings adds gravitas to the use of murine models for the study of motile cilia in this condition.2,3

Respiratory epithelium from patients with BBS tended to demonstrate an increase in cellular spaces, epithelial blebbing, and reduced ciliation. There was also an increase in the number of mucus cells present. These changes probably represented asthma and rhinitis in these patients. Patient selection for nasal biopsy based on a positive clinical history of respiratory disease, rhinitis, and reduced NO may have influenced the prevalence with which these features were seen in this cohort. Alternatively, it is possible that these changes may represent a role for BBS proteins in epithelial cell turnover and differentiation, because it has been shown that some BBS proteins have a role in signaling pathways such as Wnt signaling.17

There was double the prevalence of GP-diagnosed asthma in patients with BBS compared with the general population. At a cellular level, findings from the nasal epithelium of patients with BBS were similar to those of the cohort with asthma. Cell counts from this group are in keeping with findings from studies of bronchial epithelium in severe asthmatics who also had an increased incidence of epithelial damage and mucus cell hyperplasia.18 Interestingly, murine models of BBS show no increase in airway hyperresponsiveness and no predisposition to asthma-like responses. In this patient cohort, the similarity in epithelial changes in patients with BBS and asthma and the high prevalence of GP-diagnosed asthma suggest that there is a discrepancy between patients and the animal disease model. It is possible that the increased diagnostic frequency of asthma we see is related to the increased incidence of obesity in this cohort.

By screening a large cohort of patients with BBS, we found no clear evidence of motile ciliary dysfunction. One of the cardinal manifestations of motile cilia dysfunction is the development of permanent lung damage in the form of bronchiectasis. There was no reported increase in the number of respiratory infections per year, no report of productive cough, and no record that patients with BBS went on to develop bronchiectasis. We did not feel that CT scans to exclude bronchiectasis were justified, given the radiation associated with this investigation. However, there are some case reports of bronchiectasis in patients with ciliopathies affecting primary cilia.19 Given the diversity of BBS genotypes and mutations, it is possible that dysfunction of motile cilia will be present in a small number of patients.

Conductive hearing loss caused by otitis media is a recognized manifestation of BBS, and insertion of grommets is commonplace.2 In the current study, this occurred in 33% of patients compared with 74% of patients with PCD. Unlike patients with PCD, in whom grommets often discharge, the majority of patients with BBS reported successful outcome from the surgery. This makes it more likely that glue ear in BBS is related to another factor, such as altered maxillofacial morphology, rather than motile cilia dysfunction.

In conclusion, motile ciliary structure and function were essentially normal in > 90% of cilia sampled in this BBS cohort. Patients do not have the classic clinical phenotype associated with motile ciliary dysfunction and, as a result, do not warrant associated therapies such as chest physiotherapy or prolonged antibiotic treatment for respiratory symptoms. However, we would recommend monitoring and control of asthmatic and ear, nose, and throat symptoms. When abnormalities occur, they appear to be associated with cellular changes, such as a decrease in cilia number and goblet cell hyperplasia. Unique inclusions are seen within the ciliary membrane by electron microscopy. Investigation into the nature of the cellular changes and the vesicular inclusions by immunofluorescent techniques may advance our knowledge of the function of BBS proteins within the airways.

Author contributions: A. S. is guarantor of the article and takes responsibility for the content of the manuscript, including the data and analysis. A. S., P. L. B., and C. L. H. contributed to the study conception and design; A. S., M. D., and P. L. B. contributed to the acquisition and analysis of the data; and A. S., M. D., P. L. B., and C. L. H. contributed to the interpretation of the data and the writing and editing of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Other contributions: We thank National Health Service specialist commissioning for their continued support of both the PCD and BBS services in the United Kingdom. We also thank Tom Burgoyne, PhD, and Paul Griffin for their role in preparing samples for electron microscopy.

Additional information: The e-Tables and Videos can be found in the Supplemental Materials and Multimedia sections of the online article.

BBS

Bardet Biedl Syndrome

GP

general practitioner

NO

nitric oxide

PCD

primary ciliary dyskinesia

ppb

parts per billion

Forsythe E, Beales PL. Bardet-Biedl syndrome. Eur J Hum Genet. 2013;21(1):8-13. [CrossRef] [PubMed]
 
Shah AS, Farmen SL, Moninger TO, et al. Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia. Proc Natl Acad Sci U S A. 2008;105(9):3380-3385. [CrossRef] [PubMed]
 
Davis RE, Swiderski RE, Rahmouni K, et al. A knockin mouse model of the Bardet-Biedl syndrome 1 M390R mutation has cilia defects, ventriculomegaly, retinopathy, and obesity. Proc Natl Acad Sci U S A. 2007;104(49):19422-19427. [CrossRef] [PubMed]
 
Bush A, Cole P, Hariri M, et al. Primary ciliary dyskinesia: diagnosis and standards of care. Eur Respir J. 1998;12(4):982-988. [CrossRef] [PubMed]
 
Moalem S, Keating S, Shannon P, et al. Broadening the ciliopathy spectrum: motile cilia dyskinesia, and nephronophthisis associated with a previously unreported homozygous mutation in the INVS/NPHP2 gene. Am J Med Genet A. 2013;161A(7):1792-1796. [CrossRef] [PubMed]
 
Bukowy-Bieryłło Z, Ziętkiewicz E, Loges NT, et al. RPGR mutations might cause reduced orientation of respiratory cilia. Pediatr Pulmonol. 2013;48(4):352-363. [CrossRef] [PubMed]
 
Barbato A, Frischer T, Kuehni CE, et al. Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children. Eur Respir J. 2009;34(6):1264-1276. [CrossRef] [PubMed]
 
Chilvers MA, Rutman A, O’Callaghan C. Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia. J Allergy Clin Immunol. 2003;112(3):518-524. [CrossRef] [PubMed]
 
Shoemark A, Dixon M, Corrin B, Dewar A. Twenty-year review of quantitative transmission electron microscopy for the diagnosis of primary ciliary dyskinesia. J Clin Pathol. 2012;65(3):267-271. [CrossRef] [PubMed]
 
Smith GC, Wood AM, White IR, Pell JP, Cameron AD, Dobbie R. Neonatal respiratory morbidity at term and the risk of childhood asthma. Arch Dis Child. 2004;89(10):956-960. [CrossRef] [PubMed]
 
Shields MD, Bush A, Everard ML, McKenzie S, Primhak R; British Thoracic Society Cough Guideline Group. BTS guidelines: recommendations for the assessment and management of cough in children. Thorax. 2008;63(suppl 3):iii1-iii15. [PubMed]
 
Morice AH, McGarvey L, Pavord I; British Thoracic Society Cough Guideline Group. Recommendations for the management of cough in adults. Thorax. 2006;61(suppl 1):i1-i24. [CrossRef] [PubMed]
 
Asthma UK website. http://www.asthma.org.uk. Accessed September 30, 2013.
 
Scadding G. K., Durham S. R., Mirakian R., Jones N. S., et al. BSACI guidelines for the management of allergic and non-allergic rhinitis. Clin Exp Allergy. 2008;38(1):19-42. [CrossRef] [PubMed]
 
Scottish National Tariff 2006-07. ISD Scotland website. http://www.isdscotland.org/isd/files/2006_07ScotTariffs.xls. Accessed January 9, 2008.
 
Noone PG, Leigh MW, Sannuti A, et al. Primary ciliary dyskinesia: diagnostic and phenotypic features. Am J Respir Crit Care Med. 2004;169(4):459-467. [CrossRef] [PubMed]
 
Wiens CJ, Tong Y, Esmail MA, et al. Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling. J Biol Chem. 2010;285(21):16218-16230. [CrossRef] [PubMed]
 
Thomas B, Rutman A, Hirst RA, et al. Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J Allergy Clin Immunol. 2010;126(4):722-729, e2. [CrossRef] [PubMed]
 
Kim KH, Song BG, Park MJ, et al. Noncompaction of the myocardium coexistent with bronchiectasis and polycystic kidney disease. Heart Lung Circ. 2013;22(4):312-314. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1 –  Epithelial cell differentiation. A and B, Patients with BBS and asthma had fewer ciliated epithelial cells and more mucus cells compared with healthy control subjects and patients with PCD. D-F, Low-magnification electron micrographs of nasal epithelial strips from patients with BBS, demonstrating (D) intercellular spaces and (D and E) blebbing and goblet cell hyperplasia. C and F, When ciliated cells were present, they were often depleted of cilia. Short cilia in F probably represent regeneration. G and H, Healthy, well-ciliated epithelial strips for comparison. Scale bar = 10 μm. BBS = Bardet Biedl syndrome; PCD = primary ciliary dyskinesia.Grahic Jump Location
Figure Jump LinkFigure 2 –  Inclusions within the ciliary membrane in respiratory motile cilia from patients with BBS. A, At low magnification, inclusions are indicated by arrows. A insert, D, and E, At higher magnification, it can be seen in cross section that inclusions vary in their number, size, and electron density. F, These structures were also seen in longitudinal section within the cilium. B, Visually similar structures were observed within the cytoplasm of ciliated epithelial cells at low magnification indicated by black arrows and at higher magnification (B insert). The white arrow indicates granular bodies associated with ciliary regeneration, frequently seen in these samples. C, Axonemal structure appeared to be normal in patients with BBS. In this example, both dynein arms, radial spokes, and nexin dynein regulatory complex are present. Scale bars represent 500 nm in low-magnification images (A and B) and 100 nm in images C and F. See Figure 1 legend for expansion of abbreviation.Grahic Jump Location

Tables

Table Graphic Jump Location
TABLE 1 ]  Prevalence of Symptoms Associated With Motile Ciliary Dysfunction in Patients With BBS Compared With the General UK Population and Patients With PCD1016

Data are presented as % unless indicated otherwise. BBS = Bardet Biedl Syndrome; ENT = ear, nose, and throat; GP = general practitioner; NA = not available; PCD = primary ciliary dyskinesia.

Video 1.

Normal Ciliary Beat in Healthy Control

Video 2.

Normal Ciliary Beat in BBS

Video 3.

Ciliary Depletion and Short Cilia in BBS

References

Forsythe E, Beales PL. Bardet-Biedl syndrome. Eur J Hum Genet. 2013;21(1):8-13. [CrossRef] [PubMed]
 
Shah AS, Farmen SL, Moninger TO, et al. Loss of Bardet-Biedl syndrome proteins alters the morphology and function of motile cilia in airway epithelia. Proc Natl Acad Sci U S A. 2008;105(9):3380-3385. [CrossRef] [PubMed]
 
Davis RE, Swiderski RE, Rahmouni K, et al. A knockin mouse model of the Bardet-Biedl syndrome 1 M390R mutation has cilia defects, ventriculomegaly, retinopathy, and obesity. Proc Natl Acad Sci U S A. 2007;104(49):19422-19427. [CrossRef] [PubMed]
 
Bush A, Cole P, Hariri M, et al. Primary ciliary dyskinesia: diagnosis and standards of care. Eur Respir J. 1998;12(4):982-988. [CrossRef] [PubMed]
 
Moalem S, Keating S, Shannon P, et al. Broadening the ciliopathy spectrum: motile cilia dyskinesia, and nephronophthisis associated with a previously unreported homozygous mutation in the INVS/NPHP2 gene. Am J Med Genet A. 2013;161A(7):1792-1796. [CrossRef] [PubMed]
 
Bukowy-Bieryłło Z, Ziętkiewicz E, Loges NT, et al. RPGR mutations might cause reduced orientation of respiratory cilia. Pediatr Pulmonol. 2013;48(4):352-363. [CrossRef] [PubMed]
 
Barbato A, Frischer T, Kuehni CE, et al. Primary ciliary dyskinesia: a consensus statement on diagnostic and treatment approaches in children. Eur Respir J. 2009;34(6):1264-1276. [CrossRef] [PubMed]
 
Chilvers MA, Rutman A, O’Callaghan C. Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia. J Allergy Clin Immunol. 2003;112(3):518-524. [CrossRef] [PubMed]
 
Shoemark A, Dixon M, Corrin B, Dewar A. Twenty-year review of quantitative transmission electron microscopy for the diagnosis of primary ciliary dyskinesia. J Clin Pathol. 2012;65(3):267-271. [CrossRef] [PubMed]
 
Smith GC, Wood AM, White IR, Pell JP, Cameron AD, Dobbie R. Neonatal respiratory morbidity at term and the risk of childhood asthma. Arch Dis Child. 2004;89(10):956-960. [CrossRef] [PubMed]
 
Shields MD, Bush A, Everard ML, McKenzie S, Primhak R; British Thoracic Society Cough Guideline Group. BTS guidelines: recommendations for the assessment and management of cough in children. Thorax. 2008;63(suppl 3):iii1-iii15. [PubMed]
 
Morice AH, McGarvey L, Pavord I; British Thoracic Society Cough Guideline Group. Recommendations for the management of cough in adults. Thorax. 2006;61(suppl 1):i1-i24. [CrossRef] [PubMed]
 
Asthma UK website. http://www.asthma.org.uk. Accessed September 30, 2013.
 
Scadding G. K., Durham S. R., Mirakian R., Jones N. S., et al. BSACI guidelines for the management of allergic and non-allergic rhinitis. Clin Exp Allergy. 2008;38(1):19-42. [CrossRef] [PubMed]
 
Scottish National Tariff 2006-07. ISD Scotland website. http://www.isdscotland.org/isd/files/2006_07ScotTariffs.xls. Accessed January 9, 2008.
 
Noone PG, Leigh MW, Sannuti A, et al. Primary ciliary dyskinesia: diagnostic and phenotypic features. Am J Respir Crit Care Med. 2004;169(4):459-467. [CrossRef] [PubMed]
 
Wiens CJ, Tong Y, Esmail MA, et al. Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling. J Biol Chem. 2010;285(21):16218-16230. [CrossRef] [PubMed]
 
Thomas B, Rutman A, Hirst RA, et al. Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J Allergy Clin Immunol. 2010;126(4):722-729, e2. [CrossRef] [PubMed]
 
Kim KH, Song BG, Park MJ, et al. Noncompaction of the myocardium coexistent with bronchiectasis and polycystic kidney disease. Heart Lung Circ. 2013;22(4):312-314. [CrossRef] [PubMed]
 
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