SUBMITTED 6 OCT 21 1 REVISIONS REQ. 13 DEC 21 & 27 FEB 22; REVISIONS RECD. 30 JAN & 9 MAR 22 2 ACCEPTED 22 MAR 22 3 ONLINE-FIRST: APRIL 2022 4 DOI: https://doi.org/10.18295/squmj.4.2022.029 5 6 Cilia Ultrastructure Associated with Primary Ciliary Dyskinesia in Omani 7 Patients 8 *Kawther Al Adawi,1 Taher Baomar,2 Marwa Al Riyami,1 Nawal Al 9 Shamli,3 Khoula Al Shidhani,4 Aliya Al Ansari,2 Hussein Al Kindi3 10 11 1Department of Pathology, Sultan Qaboos University Hospital, Muscat Oman; 2Department 12 of Biology, College of Science and 3Department of Child Health, College of Medicine & 13 Health Sciences, Sultan Qaboos University, Muscat, Oman; 4Department of Child Health, 14 The Royal Hospital, Ministry of Health, Muscat, Oman 15 *Corresponding Author’s e-mail: kawthara@squ.edu.om 16 17 Abstract 18 Objectives: Primary ciliary dyskinesia (PCD) is a disorder affecting the structure and function 19 of motile cilia. Transmission electron microscopy is one method that can be used to examine 20 ciliary ultrastructure in airway biopsies. Although the role of ultrastructural findings in PCD 21 has been described in the literature, this role has not been well studied in the Middle East or, 22 by extension, Oman. This study aims to describe ultrastructural features in Omani patients with 23 high suspicion of PCD. Methods: This retrospective cross-sectional study included 129 24 adequate airway biopsies obtained between 2010–2020 from Omani patients suspected of 25 having PCD. Results: Ciliary ultrastructural abnormalities in our study population were outer 26 dynein arm associated with inner dynein arm defects (8%), microtubular 27 disorganisation associated with inner dynein arm defect (5%), and isolated outer dynein arm 28 defect (2%). Most of the biopsies sowed normal ultrastructure (82%). Conclusion: In Omani 29 patients suspected to have PCD, normal ultrastructure was the commonest feature. 30 Keywords: Cilia; Primary Ciliary Dyskinesia; Airway Biopsy; Transmission Electron 31 Microscopy; Ultrastructure; Oman. 32 33 mailto:kawthara@squ.edu.om Advances in Knowledge 34  Transmission electron microscopy (TEM) is a feasible diagnostic tool for primary 35 ciliary dyskinesia (PCD). 36  Normal ciliary ultrastructure features are common finding when using TEM. 37 Applications to Patient Care 38  A normal ciliary ultrastructure finding does not exclude PCD in Omani patients. 39  Other tests need to be considered, including genetic testing, if a ciliary ultrastructure 40 finding is normal. 41 42 Introduction 43 Primary ciliary dyskinesia (PCD) is a hereditary disorder affecting the structure and/or 44 function of motile cilia.1,2 PCD is particularly challenging to manage and research, and 45 diagnosis is typically delayed due to shared clinical features with other diseases, including 46 cystic fibrosis (CF), immunodeficiency, chronic pulmonary aspiration, asthma and recurrent 47 respiratory viral infection.2–4 48 49 The symptoms of PCD are initially observed in organs in which cilia motility is essential for 50 normal function and manifest in organs outside the respiratory tract as well as in sinuses and 51 the lungs.3 In the respiratory system, PCD-related mucociliary clearance impairment can 52 cause chronic wet cough, recurrent respiratory tract infections, bronchitis and 53 bronchiectasis.4–7 The effects may vary between patients but are common in that they never 54 fully resolve despite using systemic antibiotics.4 Outside the respiratory system, PCD patients 55 can suffer from fertility issues and hearing difficulties due to glue ear, and approximately 45–56 50% have situs inversus.5,7,8 PCD patients also can have inborn heart defects due to situs 57 ambiguuus.6 58 59 The official American Thoracic Society (ATS) clinical practice guidelines for the diagnosis 60 of PCD recommend testing for PCD if two clinical PCD phenotypes are present.4 The 61 recommended testing methodologies are the examination of ciliary ultrastructure using TEM, 62 genetic testing, nasal nitric oxide (nNO) measurement in children five years of age or older 63 and high-speed video microscopy (HSVM).3,7,8 Although HSVM is useful for accessing 64 ciliary beat frequency and its pattern and length, such testing is limited to specialised PCD 65 centres.4 66 67 Ultrastructural studies of ciliary axonemes by TEM remains one of the most widely used and 68 reliable diagnostic methods for PCD.9 Using this diagnostic process, however, is challenging, 69 because obtaining an adequate sample with a sufficient number of cilia that are technically 70 acceptable for interpretation is not easy.3,10 However, using TEM to identify a consistent 71 ultrastructural abnormality within the ciliary axoneme helps to expedite disease management 72 as it indicates a definite diagnosis.11 Ciliary ultrastructural features, including the location of 73 the central pair complex, the availability of the dynein arms, orientation of peripheral 74 microtubules (MTs), and epithelial cells abnormalities are definitive clues leading to PCD 75 diagnosis.5,10,12 76 77 International guidelines for reporting PCD using TEM were established to regulate and direct 78 the diagnostic efforts.10 According to these guidelines, ciliary ultrastructure can be classified 79 as normal, or as class 1 or class 2 defects.10 Normal ultrastructure is defined as the presence 80 of the well-known 9 + 2 axonemal structure with a clear identification of outer dynein arms 81 (ODA), inner dynein arms (IDA) and the central microtubules in the middle of the axoneme 82 [Figure 1].10 Class 1 findings are considered as hallmark defects ( i.e. diagnostic) while class 2 83 defects may possibly be used to indicate a diagnosis of PCD if it is consistent across multiple 84 samples.10 In this case, and if clinical symptoms are persistent, it is required to confirm the 85 diagnosis using another mode of testing like for example high-speed video microscopy or 86 genetic testing.10 87 88 Class 1, or hallmark defects, can include isolated loss of ODA or combined ODA and IDA 89 absence from > 50% of cross-sections. However, when it is < 50 % ( i.e. 20 -50%) it is 90 referred to as class 2 defects. In addition, microtubular disorganisation combined with IDA 91 defects is considered a class 1 defect, while microtubular disorganisation when IDA is present 92 is referred to as class 2 defect. .10 Moreover, central complex defect and the Mislocalisation of 93 basal bodies with few or no cilia are also considered class 2 defects.10 94 95 PCD is no longer considered a mild disease, and more research is needed to expedite PCD 96 management in order to prevent complications from the disease. The objective of this study, 97 therefore, was to determine the most common ciliary ultrastructural defects in Omani PCD 98 patients and use those results to assist in patient management. 99 100 All airway biopsies sent to the Electron Microscopy Unit (EMU) at Sultan Qaboos University 101 (SQU) from 2010–2020 were included in this study. This sample included biopsies from all 102 Omani patients who were highly suspected of having PCD based on clinical phenotypes and 103 symptoms. Specimens were taken from patients between one month and 70 years of age. 104 These patients had presented to clinics suffering from at least two out of four ATS-defined 105 PCD symptoms. These symptoms included recurrent chest infections; wet, productive cough; 106 the presence of laterality defects and neonatal respiratory distress.4 107 108 Methods 109 Medical ethics approval was obtained to include all airway biopsies for ciliary ultrastructural 110 examination from 2010–2020 in SQU’s EMU. The research was approved by the Medical 111 Research Ethics Committee (MREC), College of Medicine and Health Sciences at Sultan 112 Qaboos University (MREC #2089) and the Scientific Research Committee (SRC) at the 113 Royal Hospital, Ministry of Health, Sultanate of Oman (SRC #23/2020). 114 115 Araldite blocks from samples of patients attending pulmonary clinics at Sultan Qaboos 116 University Hospital (SQUH) and the Royal Hospital (RH) that had been received in the 117 EMU, Department of Pathology at SQU were collected retrospectively. In addition, two 118 adequate normal control samples were obtained from two healthy adult candidates. 119 120 Samples were considered adequate if 50 cross cilia were possible to screen using TEM. 121 Samples of adequate airway biopsies from highly suspected PCD Omani patients were all 122 included if they met the inclusion criteria. They were included if patients had presented with 123 at least two of the following symptoms: recurrent chest infections with no response to 124 antibiotics; laterality defects; respiratory distress during early infancy, year-round wet cough. 125 126 All inadequate samples were excluded as were samples from patients diagnosed with 127 conditions other than PCD after reviewing patients’ clinical charts. 128 129 Samples had been collected using the following steps: nasal airway biopsies were taken in 130 outpatient clinics. The clinicians obtained the specimens by scraping the nasal inferior 131 turbinate using either a brush or rhino pro curette. Specimens were received in Karnovsky’s 132 fixative and then transferred into sodium cacodylate buffer and kept at 4° C. Specimens were 133 then fixed in osmium tetroxide, washed in distilled water and dehydrated in a series of graded 134 acetone. The dehydrated specimens were infiltrated in a mixture of acetone and araldite resin, 135 embedded in freshly prepared pure araldite resin and polymerised at 60° C overnight. Control 136 specimens were processed following the same protocol that was used for the retrospectively 137 collected specimens. 138 139 The blocks containing ciliated cells were cut using a diamond knife, and thin sections were 140 placed on copper grids. Sections were stained using supersaturated uranyl acetate and 141 Reynolds’ lead citrate. In total, 50 cross cilia from each sample were screened at high 142 magnification using a JEOL JEM-1230 TEM at 80 KV (JEOL, Ltd. Tokyo, Japan). Images 143 were captured using a Gatan MSC SI003 1 digital camera system (Gatan, Inc., Pleasanton, 144 California, USA) and analysed. 145 146 Results 147 A total of 421 airway biopsies were received and processed during the study period, out of 148 which 129 biopsies (30%) were adequate. From the adequate samples, 114 were from 149 individuals between 1 month and 18 years old, and only 15 were from patients above 18 150 years old. These samples were of patients from different regions in the country. A sample 151 was considered adequate when 50 cross cilia could be examined and photographed with a 152 TEM. 153 154 Image analysis was done following ATS international guidelines for reporting PCD using 155 TEM. 10 Out of the 129 adequate samples, 23 (18%) showed alterations in the ciliary 156 ultrastructure [Table 1]. The absence of ODA and IDA was the most frequently observed 157 abnormality in the studied group (n = 10; 8%) [Figure 2] followed by the microtubular 158 disorganisation associated with an IDA defect (n = 6; 5%). Both of these abnormalities are 159 considered class 1 defects. The least common class 1 ultrastructural defects were the isolated 160 absence of ODA (n = 3; 2%) [Figure 3]. Additionally, 3 samples (n = 3; 2%) showed central 161 complex defects [Figure 4] and one sample (n = 1) showed microtubular disorganisation 162 without IDA defect. These types of defects are classified as class 2 defects and would require 163 another mode of testing (e.g., testing for genetic mutations) to confirm PCD diagnosis. 164 165 Most of the sample (n = 106; 82%) showed normal ultrastructure of the ciliary axoneme 166 [Figure 5]. On review, it was found that all of these individuals had fulfilled the ATS clinical 167 criteria for testing. PCD was highly suspected due to their presentation with at least two out 168 of four PCD clinical phenotypes. Furthermore, 65 patients (50%) from this group had 169 negative sweat chloride test, and 57 (44%) had a negative workup for immunodeficiency. 170 However, 37 patients (29%) were not tested for immunodeficiency or sweat chloride for 171 clinical reasons. 172 173 Discussion 174 Ciliary ultrastructure analysis requires special expertise. Analysts require knowledge of 175 normal versus abnormal ciliary structures and TEM availability.13 It is important that 176 healthcare institutions overcome these challenges, however, because this type of analysis is 177 essential to the process of diagnosing PCD.8,9 TEM is feasible in about 70% of PCD patients, 178 but TEM alone is not sufficient to achieve reliable diagnosis.14 In this study, most of the 179 current studied sample showed normal ultrastructure on TEM (n = 106; 82%). Other 180 researchers have achieved similar findings.7,13,14 In their study, Papon et al. found that more 181 than half of their PCD-positive sample showed normal ultrastructure.14 182 183 Other researchers’ findings and those of the current study suggest the potential role of gene 184 mutations in causing normal ciliary ultrastructure.7,11,15–17 DNAH11 gene mutations, for 185 example, have been found to cause PCD but are associated with normal ciliary 186 ultrastructure.16 These mutations affect the structural proteins and subsequently the function 187 of ODA, but the structure still looks normal through a TEM.16 The HYDIN autosomal 188 recessive gene mutation is also associated with normal ultrastructure.18 This gene is involved 189 in the production of proteins for the central pair complex.18 190 191 Due to high rates of consanguinity in Oman, it is expected that certain PCD-associated genes 192 are predominant in the Omani population. If the most common PCD-associated gene 193 mutations in this region are associated with normal ciliary ultrastructure, then this may 194 explain the current study results. Genetic testing, however, is needed to confirm this 195 theory. In Omani cases of suspected PCD, it is recommended to re-evaluate clinical 196 phenotypes in individuals who show repetitive normal ciliary ultrastructure. If symptoms of 197 PCD persist, then other diagnostic investigations are highly recommended to confirm PCD. A 198 similar recommendation should be followed if ultrastructural features suggest class 2 defects. 199 As with class 1 defects, a final diagnosis of class 2 defects requires confirmation of disease 200 by applying, for example, genetic testing.10 201 202 Defects of ODA and IDA and microtubular disorganisation combined with IDA defects were 203 among the ciliary ultrastructural abnormalities reported in the current study. Both of which 204 are considered class one defects and confirm PCD.10 In the current study, ODA associated 205 with an IDA defect was reported in 10 patients (8%), and microtubular disorganisation 206 associated with an IDA defect was reported in seven patients (5%). On the other hand, an 207 isolated ODA defect was reported in only 2% (n = 3) of the studied group. In these cases, 208 PCD diagnosis was confirmed by TEM, and testing for gene mutations causing these 209 abnormalities become an option but were not a priority.10 210 211 The high inadequacy rate of samples submitted for TEM analysis was a challenge in the 212 current study. However, inadequacy might also indicate a specific cause of PCD. It is now 213 well known that if multiple specimens from the same patient all show a low percentage of or 214 no cilia in the epithelial cells, then it may indicate specific PCD gene mutations.18 This type 215 of finding suggests that something is not right in the ciliogenesis process. In such cases, re-216 evaluating the clinical presentation is highly recommended. If symptoms persist with no other 217 explanation, then genetic testing for PCD to explore certain gene mutations as in the protein 218 coding CCNO or MCIDAS genes may be considered.18 Future research should examine 219 possible genetic mutations in Omani PCD patients and correlate them with the clinical and 220 ultrastructural phenotypes of patients. 221 222 Conclusion 223 The current research group recommends ciliary ultrastructural studies for PCD patients in 224 Oman. If class 1 defects are identified, early PCD management might limit or even prevent 225 lung damage due to disease complications. In this case, genetic testing is optional and may 226 not be necessary unless it is required for family planning. The percentage of cases diagnosed 227 using TEM is not high, but TEM cannot exclude PCD upon normal ultrastructure findings 228 nor when multiple specimen inadequacy is observed within the same patient. At this time, a 229 combination of tests are required to confirm PCD including TEM, genetic testing, nNO and 230 HSVM. 231 232 Conflict of Interest 233 The authors declare no conflicts of interest. 234 235 236 Funding 237 No funding was received for this study. 238 239 Acknowledgment 240 The authors would like to acknowledge the electron microscopy team at SQU for their help in 241 providing the specimens and equipment used in this research and in special Ms. Marwa Al 242 Shukri for the great help in producing the diagrams of the abnormal cilia. 243 244 Authors’ Contribution 245 KAA Carried out the electron microscopy laboratory work related to this research and 246 prepared the samples. Then screened them, captured the images and analyzed the results of 247 the research under the supervision of the team. TB was the main supervisor for this research 248 and reviewed the ultrastructure of cilia and helped in the results analysis. MAR authorized 249 the final reports of the ultrastructure for the patients included in this research. AAA and HAK 250 were the co-supervisors for this research and participated in the analysis of results. HAK is 251 also one of the clinicians who participated in the analysis of the clinical features for the 252 patients included in this research. NAS and KAS were the clinicians who reviewed the 253 clinical features and participated in the setup of this research. All authors approved the final 254 version of this manuscript. 255 256 References 257 1. Lee SL, O’Callaghan C, Lau YL, Lee CWD. Functional analysis and evaluation of 258 respiratory cilia in healthy Chinese children. Respir Res. 2020 Oct 9;21(1):259. doi: 259 10.1186/s12931-020-01506-w.. 260 2. Rubbo B, Lucas JS. Clinical care for primary ciliary dyskinesia: Current challenges and 261 future directions. Eur Respir Rev. 2017 Sep 6;26(145):170023. doi: 10.1183/16000617.0023-262 2017. 263 3. Stillwell PC, Wartchow EP, Sagel SD. Primary ciliary dyskinesia in children: A review for 264 pediatricians, allergists, and pediatric pulmonologists. . Pediatr Allergy Immunol Pulmonol. 265 2011 Dec;24(4):191-196. doi: 10.1089/ped.2011.0099. 266 4. Shapiro AJ, Davis SD, Polineni D, Manion M, Rosenfeld M, Dell SD, et al. Diagnosis of 267 primary ciliary dyskinesia: An official American thoracic society clinical practice guideline. 268 Am J Respir Crit Care Med. 2018 Jun 15;197(12):e24-e39. doi: 10.1164/rccm.201805-269 0819ST. 270 5. O’Callaghan C, Rutman A, Williams G, Kulkarni N, Hayes J, Hirst RA. Ciliated conical 271 epithelial cell protrusions point towards a diagnosis of primary ciliary dyskinesia. Respir Res. 272 2018 Jun 25;19(1):125. doi: 10.1186/s12931-018-0782-3. 273 6. Horani A, Ferkol TW, Dutcher SK, Brody SL. Genetics and biology of primary ciliary 274 dyskinesia. . Paediatr Respir Rev. 2016 Mar;18:18-24. doi: 10.1016/j.prrv.2015.09.001. 275 7. Knowles MR, Zariwala M, Leigh M. Primary Ciliary Dyskinesia. Clin Chest Med. 2016 276 Sep;37(3):449-61. doi: 10.1016/j.ccm.2016.04.008. 277 8. Tang D, Sha Y, Gao Y, Zhang J, Cheng H, Zhang J, et al. Novel variants in DNAH9 lead to 278 nonsyndromic severe asthenozoospermia. Reprod Biol Endocrinol. 2021 Feb 20;19(1):27. 279 doi: 10.1186/s12958-021-00709-0. 280 9. Thomas B, Rutman A, O’Callaghan C. Disrupted ciliated epithelium shows slower ciliary 281 beat frequency and increased dyskinesia. Eur Respir J. 2009 Aug;34(2):401-4. doi: 282 10.1183/09031936.00153308. 283 10. Shoemark A, Boon M, Brochhausen C, Bukowy-Bieryllo Z, de Santi MM, Goggin P, et al. 284 International consensus guideline for reporting transmission electron microscopy results in 285 the diagnosis of Primary Ciliary Dyskinesia (BEAT PCD TEM Criteria). Eur Respir J. 2020 286 Apr 16;55(4):1900725. doi: 10.1183/13993003.00725-2019. 287 11. Olin JT, Burns K, Carson JL, Metjian H, Atkinson JJ, Davis SD, et al. Diagnostic yield of 288 nasal scrape biopsies in primary ciliary dyskinesia: A multicenter experience. Pediatr 289 Pulmonol. 2011 May;46(5):483-8. doi: 10.1002/ppul.21402. 290 12. Li F-Q, Siller SS, Takemaru K-I. Oncotarget Basal body docking in airway ciliated cells. 291 Oncotarget. 2015 Aug 21;6(24):19944-5. doi: 10.18632/oncotarget.4609. 292 13. Haarman EG, Schmidts M. Accuracy of diagnostic testing in primary ciliary dyskinesia: Are 293 we there yet? Eur Respir J. 2016 Mar;47(3):699-701. doi: 10.1183/13993003.01914-2015. 294 14. Papon JF, Coste A, Roudot-Thoraval F, Boucherat M, Roger G, Tamalet A, et al. A 20-year 295 experience of electron microscopy in the diagnosis of primary ciliary dyskinesia. Eur Respir 296 J. 2010 May;35(5):1057-63. doi: 10.1183/09031936.00046209. 297 15. Shapiro AJ, Zariwala MA, Ferkol T, Davis SD, Sagel SD, Dell SD, et al. Diagnosis, 298 monitoring, and treatment of primary ciliary dyskinesia: PCD foundation consensus 299 recommendations based on state of the art review. Pediatr Pulmonol. 2016 Feb;51(2):115-32. 300 doi: 10.1002/ppul.23304. 301 16. Knowles MR, Leigh MW, Carson JL, Davis SD, Dell SD, Ferkol TW, et al. Mutations of 302 DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure. 303 Thorax. 2012 May;67(5):433-41. doi: 10.1136/thoraxjnl-2011-200301. 304 17. Shoemark A, Burgoyne T, Kwan R, Dixon M, Patel MP, Rogers A v., et al. Primary 305 ciliary dyskinesia with normal ultrastructure: Three-dimensional tomography detects absence 306 of DNAH11. Eur Respir J. 2018 Feb 21;51(2):1701809. doi: 10.1183/13993003.01809-2017. 307 18. Shapiro AJ, Leigh MW. Value of transmission electron microscopy for primary ciliary 308 dyskinesia diagnosis in the era of molecular medicine: Genetic defects with normal and non-309 diagnostic ciliary ultrastructure. Ultrastruct Pathol. 2017 Nov-Dec;41(6):373-385. doi: 310 10.1080/01913123.2017. 311 312 Table 1: Shows the ultrastructure detected by TEM from 2010 to 2020 313 Ultrastructural defect Number of specimens Class 1 defect ODA + IDA defects 10 (8%) Microtubular disorganisation with IDA defect 7 (5%) ODA defect ) 2 (2%) Class 2 defect Central Complex defect 3 (2%) Microtubular disorganisation with IDA present 1 (1%) Normal Ultrastructure 106 (82%) Total Adequate airway biopsies 129 314 315 316 317 318 319 320 321 322 323 324 Figure 1: A schematic diagram of the cross section of a motile cilium illustrating the normal 325 9+2 microtubular structure. The 9 fused doublet microtubules with outer and inner dynein 326 arms are arranged in the outer periphery surrounding a pair of singlet microtubules in the 327 middle of the ciliary axoneme. The central pair is surrounded by a central sheath and radial 328 spokes are radiating between them and the outer doublets. Diagram was taken from: 329 Ishikawa, H., & Marshall, W. F. (2017). Intraflagellar Transport and Ciliary Dynamics. Cold 330 Spring Harb Perspect Biol. 2017; 9(3):a021998. Published 2017 Mar 1. 331 https://doi.org/10.1101/cshperspect.a021998 332 333 334 335 336 337 338 339 340 341 342 Figure 2: (A) An electron micrograph of a class 1 defect showing the absence of both ODA 343 & IDA in the 9+2 ultrastructure of the ciliary axoneme from an adequate patient’s sample. 344 (B) A schematic diagram of the cross section of a motile cilium illustrating the absence of 345 both ODA + IDA. 346 347 348 Figure 3: (A) an electron micrograph showing a class 1 defect, ODA is missing from the ciliary 349 axoneme, while IDA (black arrow) can be identified. (B) A schematic diagram of the cross 350 section of a motile cilium illustrating the absence of ODA. 351 A B A B 352 353 Figure 4: (A) Electron micrograph of a class 2 defect. The majority of the cilia in this sample 354 showed the absence of central complex (black arrows). However, ODA & IDA can be 355 identified in those cilia. (B) A schematic diagram of the cross section of a motile cilium 356 illustrating the absence of the central complex. 357 358 359 Figure 5: Electron micrograph showing normal 9+2 ultrastructure of the ciliary axoneme from 360 an adequate patient’s sample, ODA (elbow arrow), IDA (arrow) & central complex ( arrow 361 head) are identified in this image. 362 A B