Pediatric and Developmental Pathology 17, 465–469, 2014 DOI: 10.2350-14-06-1504-OA.1 ª 2014 Society for Pediatric Pathology
Ciliary Inclusion Disease: Report of a New Primary Ciliary Dyskinesia Variant ERIC P. WARTCHOW,1* RONALD JAFFE,2 1 2
AND
GARY W. MIERAU1
Children’s Hospital Colorado, Department of Pathology, Aurora, CO, USA University of Pittsburgh School of Medicine, Children’s Hospital of UPMC, Department of Pathology, Pittsburgh, PA, USA
Received June 6, 2014; accepted October 5, 2014; published online October 9, 2014.
ABSTRACT Biopsies from 6 children with clinical presentations suggestive of primary ciliary dyskinesia (PCD) displayed respiratory epithelial cells with disorganized accumulations of basal bodies within the cytoplasm and large intracytoplasmic vesicles into which projected numerous microvilli and cilia. Microvilli, but few cilia, were present at the cell surface. Ultrastructural study revealed a variety of nonspecific abnormalities but demonstrated the cilia generally to be morphologically normal, suggesting that the cause of cilia malfunction was not any recognized primary cause or secondary effect. Repeat studies from 2 patients produced similar findings. It is proposed that this entity, termed ciliary inclusion disease, represents a variant form of PCD manifesting as a consequence of improper ciliogenesis caused by inhibited cytoskeletonregulated migration of basal bodies to the luminal surface of the airway respiratory epithelial cells. Key words: ciliogenesis, PCD, primary ciliary dyskinesia
INTRODUCTION Primary ciliary dyskinesia (PCD) describes a heterogeneous group of disorders affecting the airways, in which malformed cilia contribute to improper mucociliary clearance. The consequences of mucous accumulation include chronic upper and lower respiratory infections, which progress to bronchiectasis, an irreversible scarring and widening of the bronchial tree. Other clinical manifestations include neonatal respiratory distress, chronic wet daily cough, abnormal lung function, situs disorders, and fertility abnormalities (infertility resulting from immotile sperm in males and decreased fecundity and/or an increased prevalence of ectopic pregnancies in females) [1].
A preliminary report on data contained in this study was presented in poster form at the United States and Canadian Academy of Pathology Conference, San Diego, CA (2014). *Corresponding author, e-mail: [email protected]
Cilia of the airways form within respiratory epithelial cells through a process by which centrioles within the cytoplasm multiply, migrate to the apical surface of the cell, and emerge as small motile appendages. The ability of cilia to function is directly related to ciliogenesis and the assemblage of structural components within the cilium unit. A motile cilium comprises a specialized ciliary membrane surrounding a 9 + 2 microtubule arrangement in which 2 central microtubules are encircled by 9 microtubule doublets. Movement of a single cilium is conferred by the sliding of microtubule doublets against their adjacent doublets through adenosine triphosphate hydrolysis by dynein proteins, which are attached as ‘‘arms’’ along the length of the inner and outer aspects of each microtubule doublet. Normal ciliary motion results from the synchronous bending of individual cilia en masse in a consistent pattern and frequency. A single cell may have 200 or more cilia that beat in a concerted waveform pattern to move the protective mucus layer from the distal to the proximal airway. The mucus then ascends the trachea, enters the pharynx, and is swallowed before being removed by the gastrointestinal tract, thereby effectively eliminating inhaled pathogens and toxins from the airways [2]. Primary ciliary dyskinesia occurs when a person is born without the genetic capacity to form functional cilia. These alterations to the normal genetic code occur as heterogeneous autosomal recessive mutations [1]. The diagnosis of disease 1st involves the suspicion of PCD based on the clinical phenotype and the exclusion of other, more common disease processes. Attempts have been made to standardize the current diagnostic amalgam into a logical and straightforward algorithm. However, very few centers have the ability to perform all of the components of the proposed screening and testing procedures, which include employment of high-speed video recording for analysis of ciliary beat patterns and frequencies, nasal nitric oxide testing, and immunofluorescence studies [3–5]. As a result, these studies are often omitted in favor of a tissue biopsy for ultrastructural studies. The morphological evaluation of ciliary structural components requires the use of transmission electron
Table 1.
Clinicopathologic features
Case
Sex
Age
Biopsy site
Biopsy technique
Situs disorder
1
M M
3 4 5 6
M F F F
Nasal Nasal Nasal Trachea Nasal Adenoid Trachea Trachea
Forceps Forceps Brush Forceps Brush Forceps Brush Brush
Yes
2
2y 6y 11 mo 12 mo 16 mo 13 mo 5y 11 mo
No Unknown Unknown Unknown Yes
M indicates male; F, female.
microscopy (TEM). Electron microscopy was involved in the initial description of the disease in 1976 and has enjoyed a long-standing history as the ‘‘gold standard’’ for diagnosis [6]. Identification of ultrastructural defects remains the most common and most reliable diagnostic test. Recent advances in molecular diagnostics have revealed that a substantial percentage of PCD patients display normal ciliary ultrastructure [1]. However, with the exception of mutations within a single known gene, all genetic changes associated with PCD can be attributed to specific corresponding and consistently observed ultrastructural abnormalities within individual mature cilia [7]. While it is accepted that electron microscopy cannot be relied upon to confirm the absence of PCD, it is the demonstration of such defects that remains the cornerstone of diagnosis. To date, all PCD-related ciliary dysfunction confirmed by electron microscopy has involved the abnormal structure of mature cilia. During routine examination of cilia in the TEM, the electron microscopist must search for cilia projecting from the apical surface of the epithelial cell and locate cilia oriented in cross section in order to identify the relevant structures. Therefore, little attention is generally given to the cytoplasmic organization of the epithelial cells. While disruptions of the epithelial cell cytoskeleton have been implicated in other ciliopathies, these mechanisms have yet to be described in association with congenital airway disease. We report a heretofore unrecognized condition in which cytoskeletal disruption appears to be the cause of mucociliary clearance disease.
METHODS Respiratory epithelium from 6 children suspected by clinical findings (Table 1) of having PCD was subjected to TEM examination. Multiple biopsies were available in 2 of the cases. Patient 1 was restudied 4 years after the initial procedure. Patient 2 was resampled from a different anatomic site 2 weeks after the initial biopsy. Biopsy sites included nares, trachea, and adenoid. Specimens were acquired by various means, including nasal brushing, curette scraping, and biopsy forceps. All tissues were fixed in cacodylate buffered 2.5% glutaraldehyde and
466
E.P. WARTCHOW ET AL.
Figure 1. Electron micrograph showing the relevant ultrastructural features observed from case 1: diminished cilia at the cell surface (arrowheads), large subnuclear (Nu) intracytoplasmic inclusion within epithelial cell (arrow, inset). Note the full section thickness identified by the basement membrane (BM) near the bottom of the image and luminal cell surface at the top. Scale bar 5 4 mm.
processed according to standard protocols, which include secondary fixation with 2.0% osmium tetroxide and poststaining of sections with uranyl acetate and lead citrate.
RESULTS Six patients suspected of having PCD were discovered during routine TEM examination to exhibit none of the recognized ultrastructural features associated with this group of diseases but instead to display unusual ciliary inclusions within the respiratory epithelial cells. The inclusions were usually quite large, often occupying a major portion of the cytoplasm. They were observed at varying levels within the pseudostratified epithelium and were often far removed from the luminal surface (Figs. 1,2). In addition to microvilli and cilia, the inclusions were found to contain vesicular, finely granular, and flocculent materials not present at the luminal surface. The inclusions did not resemble autophagic vacuoles or the smaller and more numerous endosomal vesicles associated with cilia resorption [8]. Further evidence that the inclusions represent intracytoplasmic cyst-like inclusions rather than autophagic vacuoles was provided by the observations of wellformed basal bodies surrounding the inclusions and from which mature cilia were projected into the inclusions
Figure 2. Electron micrograph from case 1 showing multiple inclusions occurring at varying levels of the pseudostratified layer of respiratory epithelium (asterisks). Scale bar 5 4 mm.
(Figs. 1–4). The cilia within the inclusions, as at the cell surface, were morphologically normal or displayed only abnormalities consistent with reactive secondary changes. Many of the cells not displaying ciliary inclusions contained disorganized aggregations of centrioles and basal bodies (Fig. 4). Repeat studies showed no change in the observed characteristics (Fig. 4).
DISCUSSION The unique morphologic and associated clinical findings in the 6 described cases are strongly suggestive of representing a primary genetic, rather than a secondary acquired, condition. The characteristic identifying inclusions are shown not to represent transient surface invaginations within the respiratory epithelial cells but rather to be morphologically distinctive structures that are persistent in presence and widespread in distribution within the respiratory tract. In addition to the criteria of ‘‘permanence’’ and ‘‘universality’’ thus being satisfied [9], support for this contention is provided by the observation of situs inversus totalis in at least 2 of these children. The single patient thus far available for genetic study was found not to display a mutation in any of the 12 genes associated with PCD that are available for commercial testing but was discovered to display a single ‘‘variant of unknown significance’’ in the DNAH11 gene, this being the site of mutations producing an estimated 22% of PCD cases exhibiting morphologically normal cilia [7,10]. Since this condition first came to attention, it has been detected in 6 of 364 cilia specimens (2.5%)
examined at our diagnostic facility. Its frequency of occurrence thus seems to be similar to that of the cases caused by a hallmark dynein protein ultrastructural abnormality, which has been confirmed in 12 of our 364 (3%) suspected PCD cases during the same time frame. Although more easily recognized in specimens in which the epithelial layer remains intact, the condition can with care and caution be identified as well in the disrupted specimens produced by brushing or curettage (Fig. 5). We recommend that a deliberate search for such inclusions be undertaken whenever cilia are sparse or absent at the cell surface or when a scattering of disorganized basal bodies is observed within the cytoplasm of the epithelial cells. Ciliogenesis requires the execution of several key intracellular pathways in a precise and complex sequence involving 4 stages: centriole multiplication, centriole migration to the apical surface, elongation of cilia, and formation of basal body accessory structures. The details of this process have been described in several reviews [11–13]. A recent report [14] has described a novel recessive mutation in the CCNO gene causing reduction in the quantity of surface cilia produced by respiratory epithelial cells. Transmission electron microscopy and immunofluorescence studies suggest that the subsequent functional abnormality results from defective amplification of centrioles during the earliest stages of ciliogenesis, thus illustrating the potential impact of impaired cilia differentiation. Important to our perspective is the observation that in the cases presented, centrioles are being multiplied, docked at a membrane, and elongated as if normal. The error appears to result from a disruption in the migration of the centrioles to the apical surface of the cells. In this aberrant condition, as the basal bodies begin to elongate within the cytoplasm and project into the ciliary vesicles (small pockets that are associated with the distal portion of a basal body), the ciliary vesicles aggregate and fuse errantly to form an intracellular cyst that may provide a surrogate membrane upon which the developing cilia can dock and into which they can elongate [15]. It is suspected that the disorganized accumulation of basal bodies observed in Figure 4A represents a stage of disrupted migration prior to cilia elongation and inclusion formation. The resemblance of these findings to what is seen in other conditions known to result from genetic mutations that disrupt trafficking of intracellular components is striking [16–18]. For example, it has recently been learned that mutations in the MYO5B gene cause microvillus inclusion disease. The affected myosin V motor protein, which is involved in vesicular transport processes along actin filaments, affects epithelial cell trafficking and cell polarity by causing a disorganization of the actin cytoskeleton and mislocalization of transporter proteins [19]. The actin cytoskeleton is known to play a significant role in the migration of centrioles to their docking station at the apical surface [20,21]. An actin binding inhibitor, Cytochalasin D, has been shown
CILIARY INCLUSION DISEASE
467
Figure 3. Electron micrographs showing inclusions observed in cases 2 through 5 (A–D, respectively). Scale bars 5 3 mm.
to impede centriole migration in quail oviduct epithelial cells, causing the formation of similar intracellular inclusions [22,23]. A similar mechanism may be responsible for ciliary inclusion disease.
The formation of intracellular ciliated cysts within human respiratory epithelial cells has been previously observed; however, it has never been specifically associated with airway disease. Our observations offer
Figure 4. Electron micrographs illustrating persistence of ultrastructural findings in repeat studies. (A) Case 1; Scale bar 5 2 mm. (B) Case 2; Scale bar 5 1 mm.
468
E.P. WARTCHOW ET AL.
Figure 5. Respiratory epithelial cell from a brush biopsy (case 6) displaying a large ciliary inclusion containing morphologically normal cilia. Scale bar 5 4 mm.
a 1st description of a novel variant of PCD exhibiting such findings, which we have termed ciliary inclusion disease. ACKNOWLEDGMENTS The authors would like to acknowledge Janet Walpusk for making the initial observation of the ciliary inclusions and for recognizing the potential significance of the finding and Luann Goin, Janet Lieber, and Ryan Goffredi for assistance with the preparation of electron microscopic specimens. REFERENCES 1. Knowles MR, Daniels LA, Davis SD, Zariwala MA, Leigh MW. Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease. Am J Respir Crit Care Med 2013;188:913–922. 2. Fahy J, Dickey B. Airway mucous function and dysfunction. N Engl J Med 2010;363:2233–2247. 3. Leigh MW, Hazucha MJ, Chawla KK, et al. Standardizing nasal nitric oxide measurement as a test for primary ciliary dyskinesia. Ann Am Thorac Soc 2013;10:574–581.
4. Lobo LJ, Zariwala MA, Noone PG. Primary ciliary dyskinesia. Q J Med 2014;107:691–699. 5. 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;65:267–271. 6. Afzelius BA. A human syndrome caused by immotile cilia. Science 1976;193:317–319. 7. Knowles MR, Leigh MW, Carson JL, et al. Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure. Thorax 2012;67:433–441. 8. Pampliega O, Orhon I, Patel B, et al. Functional interaction between autophagy and ciliogenesis. Nature 2013;502:194–200. 9. Mierau GW, Agostini R, Beals TF, et al. The role of electron microscopy in evaluating ciliary dysfunction: report of a workshop. Ultrastruct Pathol 1992;16:245–254. 10. Ambry Genetics. Available at http://www.ambrygen.com. Accessed February 4, 2013. 11. Avasthi P, Marshall WF. Stages of ciliogenesis and regulation of ciliary length. Differentiation 2012;83:S30–S42. 12. Dawe HR, Farr H, Gull K. Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells. J Cell Sci 2007;120:7–15. 13. Garcia-Gonzalo FR, Reiter JF. Scoring a backstage pass: mechanisms of ciliogenesis and ciliary access. J Cell Biol 2012;197:697– 709. 14. Wallmeier J, Al-Mutairi D, Chen C, et al. Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia. Nat Genet 2014;46:646–652. 15. Sorokin S. Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol 1962;15:363–377. 16. Hagiwara H, Ohwada N, Fujimoto T. Intracytoplasmic lumina in human oviduct epithelium. Ultrastruct Pathol 1997;21:163–172. 17. Hagiwara H, Ohwada N, Aoki T, Takata K. Ciliogenesis and ciliary abnormalities. Med Electron Microsc 2000;33:109–114. 18. Boysen M, Reith A. Intracytoplasmic lumina with and without cilia in both normal and pathologically altered nasal mucosa. Ultrastruct Pathol 1980;1:477–485. 19. Thoeni C, Vogel GF, Tancevski I, et al. Microvillus inclusion disease: loss of myosin Vb disrupts intracellular traffic and cell polarity. Traffic 2014;15:22–42. 20. Pedersen L, Schrøder J, Satir P, Christensen S. The ciliary cytoskeleton. Comp Physiol 2012;2:779–803. 21. Antoniades I, Stylianou P, Skourides P. Making the connection: ciliary adhesion complexes anchor basal bodies to the actin cytoskeleton. Dev Cell 2014;28:70–80. 22. Boisvieux-Ulrich E, Laine´ MC, Sandoz D. Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium. Cell Tissue Res 1990;259:443–454. 23. Schliwa M. Action of Cytochalasin D on cytoskeletal networks. J Cell Biol 1992;92:79–91.
CILIARY INCLUSION DISEASE
469
Ciliary Inclusion Disease: Report of a New Primary Ciliary Dyskinesia Variant ERIC P. WARTCHOW,1* RONALD JAFFE,2 1 2
AND
GARY W. MIERAU1
Children’s Hospital Colorado, Department of Pathology, Aurora, CO, USA University of Pittsburgh School of Medicine, Children’s Hospital of UPMC, Department of Pathology, Pittsburgh, PA, USA
Received June 6, 2014; accepted October 5, 2014; published online October 9, 2014.
ABSTRACT Biopsies from 6 children with clinical presentations suggestive of primary ciliary dyskinesia (PCD) displayed respiratory epithelial cells with disorganized accumulations of basal bodies within the cytoplasm and large intracytoplasmic vesicles into which projected numerous microvilli and cilia. Microvilli, but few cilia, were present at the cell surface. Ultrastructural study revealed a variety of nonspecific abnormalities but demonstrated the cilia generally to be morphologically normal, suggesting that the cause of cilia malfunction was not any recognized primary cause or secondary effect. Repeat studies from 2 patients produced similar findings. It is proposed that this entity, termed ciliary inclusion disease, represents a variant form of PCD manifesting as a consequence of improper ciliogenesis caused by inhibited cytoskeletonregulated migration of basal bodies to the luminal surface of the airway respiratory epithelial cells. Key words: ciliogenesis, PCD, primary ciliary dyskinesia
INTRODUCTION Primary ciliary dyskinesia (PCD) describes a heterogeneous group of disorders affecting the airways, in which malformed cilia contribute to improper mucociliary clearance. The consequences of mucous accumulation include chronic upper and lower respiratory infections, which progress to bronchiectasis, an irreversible scarring and widening of the bronchial tree. Other clinical manifestations include neonatal respiratory distress, chronic wet daily cough, abnormal lung function, situs disorders, and fertility abnormalities (infertility resulting from immotile sperm in males and decreased fecundity and/or an increased prevalence of ectopic pregnancies in females) [1].
A preliminary report on data contained in this study was presented in poster form at the United States and Canadian Academy of Pathology Conference, San Diego, CA (2014). *Corresponding author, e-mail: [email protected]
Cilia of the airways form within respiratory epithelial cells through a process by which centrioles within the cytoplasm multiply, migrate to the apical surface of the cell, and emerge as small motile appendages. The ability of cilia to function is directly related to ciliogenesis and the assemblage of structural components within the cilium unit. A motile cilium comprises a specialized ciliary membrane surrounding a 9 + 2 microtubule arrangement in which 2 central microtubules are encircled by 9 microtubule doublets. Movement of a single cilium is conferred by the sliding of microtubule doublets against their adjacent doublets through adenosine triphosphate hydrolysis by dynein proteins, which are attached as ‘‘arms’’ along the length of the inner and outer aspects of each microtubule doublet. Normal ciliary motion results from the synchronous bending of individual cilia en masse in a consistent pattern and frequency. A single cell may have 200 or more cilia that beat in a concerted waveform pattern to move the protective mucus layer from the distal to the proximal airway. The mucus then ascends the trachea, enters the pharynx, and is swallowed before being removed by the gastrointestinal tract, thereby effectively eliminating inhaled pathogens and toxins from the airways [2]. Primary ciliary dyskinesia occurs when a person is born without the genetic capacity to form functional cilia. These alterations to the normal genetic code occur as heterogeneous autosomal recessive mutations [1]. The diagnosis of disease 1st involves the suspicion of PCD based on the clinical phenotype and the exclusion of other, more common disease processes. Attempts have been made to standardize the current diagnostic amalgam into a logical and straightforward algorithm. However, very few centers have the ability to perform all of the components of the proposed screening and testing procedures, which include employment of high-speed video recording for analysis of ciliary beat patterns and frequencies, nasal nitric oxide testing, and immunofluorescence studies [3–5]. As a result, these studies are often omitted in favor of a tissue biopsy for ultrastructural studies. The morphological evaluation of ciliary structural components requires the use of transmission electron
Table 1.
Clinicopathologic features
Case
Sex
Age
Biopsy site
Biopsy technique
Situs disorder
1
M M
3 4 5 6
M F F F
Nasal Nasal Nasal Trachea Nasal Adenoid Trachea Trachea
Forceps Forceps Brush Forceps Brush Forceps Brush Brush
Yes
2
2y 6y 11 mo 12 mo 16 mo 13 mo 5y 11 mo
No Unknown Unknown Unknown Yes
M indicates male; F, female.
microscopy (TEM). Electron microscopy was involved in the initial description of the disease in 1976 and has enjoyed a long-standing history as the ‘‘gold standard’’ for diagnosis [6]. Identification of ultrastructural defects remains the most common and most reliable diagnostic test. Recent advances in molecular diagnostics have revealed that a substantial percentage of PCD patients display normal ciliary ultrastructure [1]. However, with the exception of mutations within a single known gene, all genetic changes associated with PCD can be attributed to specific corresponding and consistently observed ultrastructural abnormalities within individual mature cilia [7]. While it is accepted that electron microscopy cannot be relied upon to confirm the absence of PCD, it is the demonstration of such defects that remains the cornerstone of diagnosis. To date, all PCD-related ciliary dysfunction confirmed by electron microscopy has involved the abnormal structure of mature cilia. During routine examination of cilia in the TEM, the electron microscopist must search for cilia projecting from the apical surface of the epithelial cell and locate cilia oriented in cross section in order to identify the relevant structures. Therefore, little attention is generally given to the cytoplasmic organization of the epithelial cells. While disruptions of the epithelial cell cytoskeleton have been implicated in other ciliopathies, these mechanisms have yet to be described in association with congenital airway disease. We report a heretofore unrecognized condition in which cytoskeletal disruption appears to be the cause of mucociliary clearance disease.
METHODS Respiratory epithelium from 6 children suspected by clinical findings (Table 1) of having PCD was subjected to TEM examination. Multiple biopsies were available in 2 of the cases. Patient 1 was restudied 4 years after the initial procedure. Patient 2 was resampled from a different anatomic site 2 weeks after the initial biopsy. Biopsy sites included nares, trachea, and adenoid. Specimens were acquired by various means, including nasal brushing, curette scraping, and biopsy forceps. All tissues were fixed in cacodylate buffered 2.5% glutaraldehyde and
466
E.P. WARTCHOW ET AL.
Figure 1. Electron micrograph showing the relevant ultrastructural features observed from case 1: diminished cilia at the cell surface (arrowheads), large subnuclear (Nu) intracytoplasmic inclusion within epithelial cell (arrow, inset). Note the full section thickness identified by the basement membrane (BM) near the bottom of the image and luminal cell surface at the top. Scale bar 5 4 mm.
processed according to standard protocols, which include secondary fixation with 2.0% osmium tetroxide and poststaining of sections with uranyl acetate and lead citrate.
RESULTS Six patients suspected of having PCD were discovered during routine TEM examination to exhibit none of the recognized ultrastructural features associated with this group of diseases but instead to display unusual ciliary inclusions within the respiratory epithelial cells. The inclusions were usually quite large, often occupying a major portion of the cytoplasm. They were observed at varying levels within the pseudostratified epithelium and were often far removed from the luminal surface (Figs. 1,2). In addition to microvilli and cilia, the inclusions were found to contain vesicular, finely granular, and flocculent materials not present at the luminal surface. The inclusions did not resemble autophagic vacuoles or the smaller and more numerous endosomal vesicles associated with cilia resorption [8]. Further evidence that the inclusions represent intracytoplasmic cyst-like inclusions rather than autophagic vacuoles was provided by the observations of wellformed basal bodies surrounding the inclusions and from which mature cilia were projected into the inclusions
Figure 2. Electron micrograph from case 1 showing multiple inclusions occurring at varying levels of the pseudostratified layer of respiratory epithelium (asterisks). Scale bar 5 4 mm.
(Figs. 1–4). The cilia within the inclusions, as at the cell surface, were morphologically normal or displayed only abnormalities consistent with reactive secondary changes. Many of the cells not displaying ciliary inclusions contained disorganized aggregations of centrioles and basal bodies (Fig. 4). Repeat studies showed no change in the observed characteristics (Fig. 4).
DISCUSSION The unique morphologic and associated clinical findings in the 6 described cases are strongly suggestive of representing a primary genetic, rather than a secondary acquired, condition. The characteristic identifying inclusions are shown not to represent transient surface invaginations within the respiratory epithelial cells but rather to be morphologically distinctive structures that are persistent in presence and widespread in distribution within the respiratory tract. In addition to the criteria of ‘‘permanence’’ and ‘‘universality’’ thus being satisfied [9], support for this contention is provided by the observation of situs inversus totalis in at least 2 of these children. The single patient thus far available for genetic study was found not to display a mutation in any of the 12 genes associated with PCD that are available for commercial testing but was discovered to display a single ‘‘variant of unknown significance’’ in the DNAH11 gene, this being the site of mutations producing an estimated 22% of PCD cases exhibiting morphologically normal cilia [7,10]. Since this condition first came to attention, it has been detected in 6 of 364 cilia specimens (2.5%)
examined at our diagnostic facility. Its frequency of occurrence thus seems to be similar to that of the cases caused by a hallmark dynein protein ultrastructural abnormality, which has been confirmed in 12 of our 364 (3%) suspected PCD cases during the same time frame. Although more easily recognized in specimens in which the epithelial layer remains intact, the condition can with care and caution be identified as well in the disrupted specimens produced by brushing or curettage (Fig. 5). We recommend that a deliberate search for such inclusions be undertaken whenever cilia are sparse or absent at the cell surface or when a scattering of disorganized basal bodies is observed within the cytoplasm of the epithelial cells. Ciliogenesis requires the execution of several key intracellular pathways in a precise and complex sequence involving 4 stages: centriole multiplication, centriole migration to the apical surface, elongation of cilia, and formation of basal body accessory structures. The details of this process have been described in several reviews [11–13]. A recent report [14] has described a novel recessive mutation in the CCNO gene causing reduction in the quantity of surface cilia produced by respiratory epithelial cells. Transmission electron microscopy and immunofluorescence studies suggest that the subsequent functional abnormality results from defective amplification of centrioles during the earliest stages of ciliogenesis, thus illustrating the potential impact of impaired cilia differentiation. Important to our perspective is the observation that in the cases presented, centrioles are being multiplied, docked at a membrane, and elongated as if normal. The error appears to result from a disruption in the migration of the centrioles to the apical surface of the cells. In this aberrant condition, as the basal bodies begin to elongate within the cytoplasm and project into the ciliary vesicles (small pockets that are associated with the distal portion of a basal body), the ciliary vesicles aggregate and fuse errantly to form an intracellular cyst that may provide a surrogate membrane upon which the developing cilia can dock and into which they can elongate [15]. It is suspected that the disorganized accumulation of basal bodies observed in Figure 4A represents a stage of disrupted migration prior to cilia elongation and inclusion formation. The resemblance of these findings to what is seen in other conditions known to result from genetic mutations that disrupt trafficking of intracellular components is striking [16–18]. For example, it has recently been learned that mutations in the MYO5B gene cause microvillus inclusion disease. The affected myosin V motor protein, which is involved in vesicular transport processes along actin filaments, affects epithelial cell trafficking and cell polarity by causing a disorganization of the actin cytoskeleton and mislocalization of transporter proteins [19]. The actin cytoskeleton is known to play a significant role in the migration of centrioles to their docking station at the apical surface [20,21]. An actin binding inhibitor, Cytochalasin D, has been shown
CILIARY INCLUSION DISEASE
467
Figure 3. Electron micrographs showing inclusions observed in cases 2 through 5 (A–D, respectively). Scale bars 5 3 mm.
to impede centriole migration in quail oviduct epithelial cells, causing the formation of similar intracellular inclusions [22,23]. A similar mechanism may be responsible for ciliary inclusion disease.
The formation of intracellular ciliated cysts within human respiratory epithelial cells has been previously observed; however, it has never been specifically associated with airway disease. Our observations offer
Figure 4. Electron micrographs illustrating persistence of ultrastructural findings in repeat studies. (A) Case 1; Scale bar 5 2 mm. (B) Case 2; Scale bar 5 1 mm.
468
E.P. WARTCHOW ET AL.
Figure 5. Respiratory epithelial cell from a brush biopsy (case 6) displaying a large ciliary inclusion containing morphologically normal cilia. Scale bar 5 4 mm.
a 1st description of a novel variant of PCD exhibiting such findings, which we have termed ciliary inclusion disease. ACKNOWLEDGMENTS The authors would like to acknowledge Janet Walpusk for making the initial observation of the ciliary inclusions and for recognizing the potential significance of the finding and Luann Goin, Janet Lieber, and Ryan Goffredi for assistance with the preparation of electron microscopic specimens. REFERENCES 1. Knowles MR, Daniels LA, Davis SD, Zariwala MA, Leigh MW. Primary ciliary dyskinesia. Recent advances in diagnostics, genetics, and characterization of clinical disease. Am J Respir Crit Care Med 2013;188:913–922. 2. Fahy J, Dickey B. Airway mucous function and dysfunction. N Engl J Med 2010;363:2233–2247. 3. Leigh MW, Hazucha MJ, Chawla KK, et al. Standardizing nasal nitric oxide measurement as a test for primary ciliary dyskinesia. Ann Am Thorac Soc 2013;10:574–581.
4. Lobo LJ, Zariwala MA, Noone PG. Primary ciliary dyskinesia. Q J Med 2014;107:691–699. 5. 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;65:267–271. 6. Afzelius BA. A human syndrome caused by immotile cilia. Science 1976;193:317–319. 7. Knowles MR, Leigh MW, Carson JL, et al. Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure. Thorax 2012;67:433–441. 8. Pampliega O, Orhon I, Patel B, et al. Functional interaction between autophagy and ciliogenesis. Nature 2013;502:194–200. 9. Mierau GW, Agostini R, Beals TF, et al. The role of electron microscopy in evaluating ciliary dysfunction: report of a workshop. Ultrastruct Pathol 1992;16:245–254. 10. Ambry Genetics. Available at http://www.ambrygen.com. Accessed February 4, 2013. 11. Avasthi P, Marshall WF. Stages of ciliogenesis and regulation of ciliary length. Differentiation 2012;83:S30–S42. 12. Dawe HR, Farr H, Gull K. Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells. J Cell Sci 2007;120:7–15. 13. Garcia-Gonzalo FR, Reiter JF. Scoring a backstage pass: mechanisms of ciliogenesis and ciliary access. J Cell Biol 2012;197:697– 709. 14. Wallmeier J, Al-Mutairi D, Chen C, et al. Mutations in CCNO result in congenital mucociliary clearance disorder with reduced generation of multiple motile cilia. Nat Genet 2014;46:646–652. 15. Sorokin S. Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J Cell Biol 1962;15:363–377. 16. Hagiwara H, Ohwada N, Fujimoto T. Intracytoplasmic lumina in human oviduct epithelium. Ultrastruct Pathol 1997;21:163–172. 17. Hagiwara H, Ohwada N, Aoki T, Takata K. Ciliogenesis and ciliary abnormalities. Med Electron Microsc 2000;33:109–114. 18. Boysen M, Reith A. Intracytoplasmic lumina with and without cilia in both normal and pathologically altered nasal mucosa. Ultrastruct Pathol 1980;1:477–485. 19. Thoeni C, Vogel GF, Tancevski I, et al. Microvillus inclusion disease: loss of myosin Vb disrupts intracellular traffic and cell polarity. Traffic 2014;15:22–42. 20. Pedersen L, Schrøder J, Satir P, Christensen S. The ciliary cytoskeleton. Comp Physiol 2012;2:779–803. 21. Antoniades I, Stylianou P, Skourides P. Making the connection: ciliary adhesion complexes anchor basal bodies to the actin cytoskeleton. Dev Cell 2014;28:70–80. 22. Boisvieux-Ulrich E, Laine´ MC, Sandoz D. Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium. Cell Tissue Res 1990;259:443–454. 23. Schliwa M. Action of Cytochalasin D on cytoskeletal networks. J Cell Biol 1992;92:79–91.
CILIARY INCLUSION DISEASE
469
