F_Boswell_4197476_B14CMScclinicalscience(cellularscience)

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Title:

CEP164 antibody can be used in nasal brush biopsy slides to

identify disrupted centriole docking in possible reduced generation

of multiple motile cilia (RGMC) cases

Faye Boswell Student number: 4197476 School of Life Science Molecular Genetics and Diagnostics QMC Supervisor: Dr. Amelia Shoemark The Royal Brompton Hospital Sydney Street, London SW3 6NP Running title: Disrupted centriole docking in possible RGMC cases

This dissertation is submitted in partial fulfilment of the project requirements for the M.Sc.: B14C MSc Clinical Science (Cellular science) May 2015 Abstract: 233 words Number of tables: 3 Main text: 4595 words Number of figures: 8 Student Declaration: I declare that all the work presented in this dissertation is my own, except where otherwise stated. Signature: ……………………………………………………………………. Date: ……………………

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CEP164 antibody can be used in nasal brush biopsy slides to identify disrupted

centriole docking in possible RGMC cases

Abstract

The project aimed to investigate if missed reduced generation of multiple motile cilia (RGMC)

cases could be identified using the current diagnostic pathway for Primary Ciliary Dyskinesia

(PCD). RGMC is a rare form of Primary Ciliary Dyskinesia characterised by the reduction of

cilia in the respiratory epithelium with recent publications showing mutations in CCNO and

MCIDAS results in reduced generation of multiple motile cilia. Staining with a centrosomal

protein antibody CEP164 using immunofluorescence microscopy was performed to establish

staining characteristics in normal patients and to compare to the staining patterns in

insufficient specimens which may be candidates for RGMC. Other techniques used included

reviewing the latest findings on RGMC, a retrospective review of patients from the PCD

diagnostic service at the Royal Brompton Hospital and identifying the frequency of

intracytoplasmic cilia in ciliated epithelial cells and epithelial strips demonstrating ciliary

aplasia using transmission electron microscopy. Immunofluorescence showed there was a

significant difference in staining pattern between normal and insufficient specimens, however

RGMC did not appear to occur commonly in patients referred for diagnosis of PCD. Normal

values have now been established for these tests in healthy and unhealthy samples against

which future patients with possible RGMC can be tested. As a result, the PCD diagnostic

service can change the diagnostic protocol to help identify possible future RGMC cases.

However distinguishing possible RGMC cases from specimens affected by secondary

influences is still not possible.

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Introduction

Cilia

Cilia are evolutionary conserved organelles. In humans there are two categories of cilia,

motile and non-motile (primary cilia). Primary cilia sense extracellular stimuli and play a role

in cellular responses to both mechanical and chemical changes. They play a role in

embryonic development and cellular homeostasis in adults. Motile cilia are able to move and

can be found at a number of different sites in the body; often there are multiple motile cilia

per cell. The respiratory epithelium contains several multiciliated cells. These are covered by

motile cilia which beat vigorously in a coordinated pattern to move inhaled particles, cell

detritus and microbes trapped in mucus towards the throat.1 All cilia are composed of a

microtubule based axoneme that is enclosed within a ciliary membrane. They arise from a

basal body, a centriolar barrel anchored to the base of the ciliary membrane by transition

fibres.2 The basal body is formed from the centrisole that is shared between the cilium and

the centrosome, where each centrosome has a mother and daughter centriole. This is

surrounded by proteinaceous pericentriolar material (PCM) where microtubules are

organised. Defects in cilia formation and ciliogenesis result in various diseases in humans

called ciliopathies.3

Ciliogenesis

The formation of the ciliary membrane at the centriole is unique as it involves the attachment

of vesicles to the non-membranous mother centriole.4 Ciliogenesis is a multi-step process

which requires the assembly of multiple soluble and membranous protein complexes. The

conversion of the mother centriole involves the basal body, derived from one of the two

centrioles that constitute the centrosome, positioned close to the plasma membrane by

docking of Golgi-derived membrane vesicles and ciliary microtubules elongate from its distal

end. The intraflagellar transport (IFT) system is then responsible for moving proteins to and

from the tip of the growing axoneme5 by anterograde and retrograde transport of cargo along

the ciliary microtubules.4 Cilia membrane biogenesis and the delivery of membrane proteins

to the cilium is coordinated by polarised vesicle trafficking under the control of conserved

GTPases of the Rab and Arf families.4 Ciliogenesis can start at the G1/G0 phase of the cell

cycle and can be subdivided into two physiologically relevant pathways referred to as intra-

and extracellular pathways, where in the extracellular pathway the mother centriole docks to

the plasma membrane.4 In multiciliated cells ciliogenesis occurs through the CD and DD

pathways, where CEP63 and CEP152 complex mediates the CD pathway and the Deup1-

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Cep152 complex and CCDC78-Cep152 complex mediates the DD pathway. The Deup1-

Cep152 complex involves functional deuterosomes which act as a centriole building platform

for centriole amplification in multiciliated cells.6

Figure 1- c shows a hypothetical model showing the CD and DD ciliogenesis pathways for

multiciliated cells a) Quiescent somatic cells use a single mature mother centriole with a

primary cilium which is lost when the cell re-enters the cell cycle b) A single primary cilium is

produced from a mature centriole c) The CD and DD pathway in multiciliated cells. The

Cep63-Cep152 complex mediates the CD pathway and the Deup1-Cep152 and CCDC78-

CEP152 complexes mediate the DD pathway d) The amplified mature centrioles migrate to

the cell surface and form multi cilia. Image taken from Tang et al 2013. 6

Basal body formation at the apical surface

Forkhead box J1 (FOXJ1) is a transcription factor involved in motile ciliated cell

differentiation and is a key regulator of the motile ciliated cell differentiation process.7 FOXJ1

regulates programmes promoting basal body docking and axoneme formation. If defective

ciliogenesis occurs, the basal body can be missing resulting in impaired mucociliary

transport. The basal body component facilitates basal docking to the apical cell membrane

through proper formation of ciliary vesicles at the distal appendage during the early stages of

ciliogensesis.8

Centrosomal proteins (Ceps) are essential in ciliogenesis, where Cep164 is required for

primary ciliary formation. The Cep164 protein is made up of 1,460 residues and

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immunofluorescent (IF) microscopy shows that Cep164 localises to the centrosome and

associates specifically with mature centrioles. 5 Cep164 is indispensable for the docking of

vesicles at the mother centriole.4 One of the major functions of Cep164 in ciliogenesis is to

recruit active TTBK2 to centrioles which triggers key events in ciliogenesis, removing certain

centrosomal proteins and recruiting IFT proteins. The loss of Cep164 leads to early defects

in ciliogenesis. Cep164 is required at an early stage of primary cilia formation for the docking

of membrane vesicles to the basal body,3 where Cep164 provides the molecular link for

connecting the mother centriole to components of the machinery that initiate ciliary

membrane biogenesis.4 Cep164 levels decrease at the M-centriole during mitoses, however

protein levels do not decrease as drastically which suggests that the diminished centrosomal

recruitment is not due to degradation of Cep164.4

Figure 2 - A model of Cep164 function during ciliogenesis taken from Schmidt et al 2012.4

Primary ciliary dyskinesia (PCD)

Primary ciliary dyskinesia (PCD) is a rare genetic disorder which has autosomal recessive

inheritance and affects approximately 1 in 20,000 of the population. It is an inclusive term for

diseases that occur as a direct result of congenital defects in cilia,9 also known as a

ciliopathy where the cilia lining the respiratory epithelium fail to beat correctly or not at all.

Ineffective mucociliary clearance causes disease of the upper and lower respiratory tract,

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presenting shortly after birth. Motile cilia are found on other epithelial surfaces such as the

fallopian tubes in females and brain ependymal therefore patients with PCD can have

numerous health problems and complications such as infertility, situs inversus where the

major organs are reversed from their normal positions and heart defects. Common

respiratory infections in PCD include chronic sinusitis, bronchitis and pneumonia which can

lead to bronchiectasis, a permanent enlargement and widening of the airways. Random

organ lateralisation results from disturbed ciliary beating of nodal cilia, perturbing the leftward

flow at the embryonic node.10 PCD is a genetically heterogeneous disorder. To date, PCD

can be caused by mutations in more than 30 genes which encode for a range of proteins

required for ciliary movement or assembly. The diagnostic pathway consists of clinical

history, measurement of nasal nitric oxide (nNO). Measurement of the gas nasal nitric oxide

(nNO) is a useful way to assess airway inflammation and is raised in patient with

inflammatory airway diseases. However nasal NO is reduced in PCD and can be used as a

screening test for the condition.13 The Royal Brompton PCD diagnostic service use <250

(ppb) as an abnormal level (pers comms RBH diagnostic service). If indicated, ciliated

epithelial cells are obtained via nasal brush biopsies, the cilia assessed for beat frequency

and waveform using light microscopy (LM). The specimen is then separated into samples for

transmission electron microscopy (TEM) and immunofluorescence (IF) and fixed accordingly.

TEM can be used to visualise the ultrastructure of the cilia, and defects can be quantified.

The recent commercial availability of antibodies to a number of ciliary proteins has meant

that IF is increasingly being used to indicate the presence or absence of key ciliary

structures.

Secondary loss of cilia

Repeated infections and inflammation of the respiratory tract can result in secondary

changes of cilia including compound cilia, numerical microtubular defects and loss of outer

membrane. It has also been shown that these secondary influences can cause disorientation

of cilia.11 Cell culture can be used for the differentiation of ciliated epithelial cells to reduce

false positive tests that mistake secondary loss of cilia for PCD; the epithelial cells are grown

in a sterile environment away from environmental factors. However this is only successful in

60% of cases. As a result patients often require a repeat nasal brush biopsy when infection

free in order to supply sufficient material for analysis at light and electron microscopy. Some

patients requiring up to 6 repeat brushings before sufficient material is available for analysis

(pers comms RBH diagnostic service).

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Mucociliary clearance disorder and ciliary aplasia

Reduced ciliary motility results in mucociliary clearance disorders such as PCD.12 A reduction

in generation of multiple motile respiratory cilia leads to recurrent infections of upper and

lower airways as mucus fails to be cleared effectively from the respiratory tract. The CCNO

gene promotes mother centriole amplification and maturation in preparation for apical

docking in ciliated cells. Mutations in the CCNO gene results in the complete absence or

marked reduction of cilia due to defective mother centriole generation and placement,

however any cilia present show no motility defects as ciliary motility proteins are still present.

As CCNO mutant cilia do not exhibit beating defects, it is distinct from PCD and does not

meet the same criteria, resulting in a new mucociliary clearance disorder on a molecular

level.1 Patients with CCNO mutations still exhibit similar symptoms as patients with PCD,

including upper and lower respiratory infections, bronchiectasis, reduced nasal nitric oxide

and possible respiratory failure.12 CCNO works in parallel to FoxJ1 protein and is expressed

in the apical cytoplasm of multiciliated cells and acts downstream of multicillin, a protein

which governs the generation of multiciliated cells by promoting early stages of cell

differentiation and is encoded by the MCIDAS gene. MCIDAS mutant respiratory epithelial

cells carry only one or two cilia per cell1 similar to CCNO mutant cells, however lack

axonemal ciliary motility proteins (such as DNAH5, CCDC39) resulting in ciliary beat defects

as seen in PCD. CCNO is absent in mutated MCIDAS cells which supports downstream

activity mentioned previously.1 Both MCIDAS and CCNO are required for deuterosomes

mediated acentrolar assembly pathway, deuterosome act as assembly sites for centriole

duplication and mother centriole assembly during multiciliated cell.12 CCNO and MCIDAS

mutations were described phenotypically as ‘cilia aplasia’ as cilia cannot be detected on

TEM,1 however this term is now outdated and incorrect as these mutations are identified as

reduced generation of multiple motile cilia (RGMC) disorder. It may be difficult to distinguish

this disorder from secondary loss of cilia as a result of an infection or inflammatory process.

Secondary loss of cilia may have been mistaken when these defects were present due to the

sometimes complete lack of cilia on the surface of respiratory epithelial cells. These defects

are predicted to be just two of a growing list of genetic defects which cause RGMC.

In the CCNO study TEM studies showed either a complete absence or severely decreased

number of cilia. Apical cell regions had normal microvilli composition but a severe decrease

of basal bodies, showing that CCNO dysfunction results in a marked reduction of centrioles.

Further analysis using an antibody to acetylated α-tubulin confirmed these TEM, findings with

1.2 cilia detected per multiciliated cell.12 In the MCIDAS study TEM findings shows reduced

cilia number and basal body mislocalisation, consistent with defective degeneration of

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multiple motile cilia (MMC). AT IF, staining with antibodies targeting acetylated α-tubulin

shows a severe reduction of MMCs compared with healthy controls and that most ciliated

cells were devoid of any cilia .1

Figure 3 - Electron Micrograph taken from Boon et al 2014 showing respiratory epithelial

cells in healthy patients (Top left) and in unhealthy patients with MCIDAS mutation (Top right

and bottom). Notice in the unhealthy samples the absence of cilia and mislocalised basal

bodies .1

PCD diagnostic service

There are three national diagnostic centres in England which diagnose PCD, however

RGMC cases may have been missed at LM and TEM analysis in the past. This may be

because potential candidates were missed during current diagnostic workup strategies since

reduced numbers of MMC can also result from secondary damage to the airways.12 RGMC

does not result in a complete absence of cilia, so where the term cilia aplasia has been used

in the past to possible describe these patients this is no longer relevant in light of confirmed

genetic mutations. To help establish a way of identifying possible RGMC cases we need to

understand the distribution of features described of these cases in normal and unhealthy

samples using CEP164 to establish if they could be used in a clinical setting to help with

identification.

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Aim

The aim of this project is to develop tools for use in the NHS to identify cases of

reduced generation of multiple motile cilia (RGMC)

The aim will be met by the following objectives:

- Patient database search to identify patients who have had repeated samples with

absent cilia

- Optimise and establish the staining pattern of CEP164 in healthy nasal columnar

epithelial cells by IF

- Compare the staining pattern in healthy patients/ insufficient patients and images of

CCNO and MCIDAS cases in the published literature

- Establish normal quantification of intracytoplasmic cilia, undocked centrioles and

nude epithelial strips by TEM for future comparison with RGMC cases

Hypothesis

Congenital mucociliary clearance disorder with reduced generation of multiple motile cilia

occurs commonly (>5% cases) in patients referred for testing for Primary Ciliary Dyskinesia.

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Methods

Patient review to identify candidates with possible congenital RGMC defects

To identify potential specimens to observe, a retrospective review of patients from the PCD

diagnostic service database at the Royal Brompton Hospital was performed. Patients were

identified who required repeat nasal brushing biopsies due to insufficient samples at LM

Nasal brushing biopsies involve the collection of ciliated epithelial cell strips from the inferior

nasal turbinates by brushing with a modified bronchoscopy cytology brush. An insufficient

sample was defined as having sparsely ciliated epithelial cells or completely nude epithelial

cells with no cilia present. Patients who required repeat brushings were separated into a list

and all other brushings associated with the patient collated together. Samples that were

insufficient for TEM were highlighted. At LM, if the observer judges that the sample does not

contain enough cilia to perform a full and adequate TEM analysis it will not be sent and

instead may be sent for cell culture and possibly IF. Potential candidates for a RGMC defect

were chosen, especially those who appeared to have nude epithelial cells or a significant

decrease in cilia in more than one brushing. Some samples were noted as containing a lot of

mucus or as ‘unhealthy’ samples with secondary defects such that an infection may be a

possible cause of a reduction in cilia, especially if the patient had no history of insufficient

samples. Reviewing the patients could help the department to assess the feasibility for using

this test for possible RGMC cases in the future.

Sample selection

‘Normal’ samples were stored nasal brush biopsies from patients who had a healthy number

of fully functioning cilia (assessment of a minimum of 6 epithelial strips) observed at LM.

Insufficient samples were unhealthy and/or nude epithelial strips at LM or TEM, which lacked

sufficient ciliated cells to make a diagnosis from. Samples with a high number of secondary

defects were samples with excess mucus and blood. Insufficient samples with a subsequent

count at TEM were specimens classed at insufficient at LM but still had enough material to

produce a count at TEM.

Sample preparation

Nasal brushings were collected into a universal containing media 199 and refrigerated until

analysis. After the sample had been analysed for ciliary beat frequency (CBF), subsequent

immunofluorescent slides were prepared by smearing the specimen onto a labelled glass

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slide, which was then air dried in a class 1 microbiological safety cabinet. When completely

dried, the slides were frozen until staining.

Immunofluorescence

IF is commonly used in the investigation or diagnosis of many diseases, where antibodies

labelled with a fluorophore are used to visualise a target protein in cells when viewed under a

fluorescent microscope.

Slides were covered with 4% paraformaldehyde fixed for 15 minutes, washed three times

with Phosphate Buffered Saline (PBS) + 0.1% Triton wash solution. 5% milk powder blocking

solution was pipetted onto the specimens and incubated for 1 hour at room temperature.

After this time, slides were washed three times with wash solution. The CEP164 antibody at

a concentration of 1:500 in PBS + 0.1% Triton was prepared and slides double labelled with

8µl of acetylated tubulin to mark the cilia. Slides were incubated for 2 hours at room

temperature. During this time the secondary antibodies were prepared by adding 5µl of both

goat anti-mouse 488 (against the acetylated tubulin) and goat anti-rabbit 594 (against

CEP164) to 5mls of PBS + 0.1% Triton. Slides were washed 3 times with wash solution and

the secondary antibodies added and allowed to incubate for 30 minutes at room temperature.

Slides were washed a further 3 times in PBS and left to air dry. Mounting media containing

DAPI was pipetted onto each slide. Slides were coverslipped and stored in the fridge in foil.

Slides were analysed using a fluorescent microscope using AxioVision 4.8 software to take

images of the cells. Immersion oil was applied to the slides and analysed using x40 (x10

objective).

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Optimising antibody staining

Before IF could be performed on patient samples, tests were performed to determine the

optimum concentration of CEP164 antibody which could produce the best visual results

whilst using the lowest possible concentration.

Figure 4 - Gallery image of nasal cilia from a healthy control, stained with CEP164 1:100,

acetylated tubulin and DAPI. Red= CEP164 (C), green= acetylated tubulin (B), Blue= DAPI

(A) and combined (D). The antibody was tested on normal controls initially with three

concentrations on the first test. At 1:100, the staining appeared to be extremely bright with

excessive background staining, which could prevent the correct analysis of CEP164 staining.

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Figure 5 - Image showing CEP164 staining of nasal cilia from a healthy control, stained with

CEP164 1:400. Red= CEP164, green= acetylated tubulin, Blue= DAPI. Staining at a

concentration of 1:400 showed reduced staining compared to the other two tests with

excellent staining of CEP164. Randomisation and blinding was employed to prevent bias.

For the second concentration test increasingly diluted CEP164 antibodies were analysed for

quality of staining to see if less of the antibody could be used for each run. CEP164 at a

concentration of 1:500 showed good staining for the antibody, equal to a concentration of

1:400.

1:800 concentration of CEP164 showed adequate staining with decreased appearance of

background staining, however with some reduction in cellular staining quality. A run was also

performed without acetylated tubulin to ascertain there was no interference between the two

primary antibodies.

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Quantifying the CEP164 staining patterns in normal and insufficient specimens using

Immunofluorescence

When analysing the pattern of staining in the ciliated epithelial cells of each specimen

particular characteristics were recorded as present or not present. Characteristics included

nuclear ‘spots’, apical surface ‘line’, gradient cytoplasmic staining, even cytoplasmic staining,

staining of the cilia and cytoplasmic staining that extends to the cilia. These categories were

identified during the first two test runs of IF as these patterns of staining were commonly

seen within samples. The aim of quantifying the staining patterns in this way was to allow

particular patterns of staining with CEP164 to be identified and to determine whether these

were characteristic of normal and insufficient samples. This may indicate what cellular

process is happening in potential RGMC cases during ciliogenesis.

Figure 6 - Ciliated nasal epithelial cells stained with CEP164 1:500. Identified characteristics

include even distribution of cytoplasmic staining (a), the appearance of a cilia ‘line’ of staining

at the base of the cilia (b), ‘granular’ staining appearance of the cytoplasm (c), the

appearance of dense spots in the nucleus (d) and staining in the nucleus (e). Red= CEP164,

green= acetylated tubulin, Blue= DAPI.

(b) (a) (d)

(c)

(e)

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Investigating level of ciliation and intracytoplasmic cilia using TEM

Specimens previously diagnosed in the PCD diagnostic service were collected from known

PCD cases, insufficient specimens, insufficient specimens with a subsequent cilia count and

specimens with a higher than average proportion of secondary ciliary defects. The

specimens were used to count the number of ciliated epithelial cells with cilia present. The

90nm sections of araldite embedded nasal brushings were analysed on a Hitachi H7000

transmission electron microscope. Ciliated epithelial cells included cells which had the basal

body of cilia present at the surface of the cell in order to observe the proportion of ‘nude’

ciliated cells compared to fully ciliated cells, as well as to observe the difference between

insufficient samples and samples which had known secondary defects. The presence of

centrioles and intracytoplasmic cilia in the cytoplasm were also identified and photographed,

which were then reviewed by an independent blinded electron micrscopist.

Ethical Considerations

The study was approved by the Hounslow and Hillingdon research ethics committee. Study

no. 07/H0709/73. Informed verbal and written consent was obtained from each of the study

participants for the use of their data and nasal brushings for this research project.

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Results

Retrospective clinical data study to identify cases of RGMC

The aim of the retrospective study of past patients in the PCD diagnostic service database

was to identify whether any possible RGMC cases could have been missed. A database of

169 referrals to the National PCD diagnostic service since commissioning began in 2006 was

analysed. The final list highlighted 11 patients with a LM description which referred to

repeated sparsely ciliated or ‘nude’ epithelia strips. The clinical results from these 11 patients

are shown below in Table 1.

Table 1 - Results from retrospective clinical data study to identify possible cases of ciliary

RGMC.

Patient Number of

brushings

containing nude

epithelial strips

Number of

‘unhealthy’

brushings

with excess

mucus/blood

Genetic

screening for

CCNO (+ve, -ve,

not performed)

Nasal NO

1 1 1 Not performed Too young for

nasal NO


nasal NO


nasal NO


nasal NO

5 1 0 Not performed 412


nasal NO


nasal NO


nasal NO

9 1 1 Not performed 9


nasal NO

11 1 0 Not performed 7.02

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After review all 11 of the patients were found to have a subsequent sufficient/healthy

brushing and were therefore not suspected as having RGMC disorder. None of the patients

had undergone genetic testing for PCD related mutations.

Immunofluorescent CEP164 staining patterns in nasal brushings with insufficient cilia

for diagnosis compared to normal controls

Table 2 – Summary of immunofluorescent CEP164 staining patterns in nasal brushings with

insufficient cilia for diagnosis compared to normal controls cell counts

No. of cells with

nuclear dot

No. of cells with

nuclear staining

No. of cells with

cytoplasmic staining

No. of cells with

gradient

No. of cells with cilia line

No. of cells with even

cytoplasmic staining

No. of cells with cilia

staining

Normal % of total 29.1 65 100 35 18.4 64.1 25.2

Insufficient % of total 12.0 17.5 100.0 14.1 15.0 87.2 23.6

% Difference 17.1 47.5 0.0 20.9 3.4 -23.1 1.6

20 ciliated cells were counted in each sample with 337 cells counted overall. 5 normal

samples and 13 insufficient samples were analysed. Some insufficient samples did not

contain up to 20 ciliated cells so less may have been counted. Overall normal specimens had

17.1% increased presence of the ‘nucleus dot’ staining characteristic compared to insufficient

samples. Normal specimens had 47.5% more ciliated cells with nuclear staining, 20.9% more

ciliated cells with ‘gradient’ pattern staining, 3.4% more ciliated cells with apical surface line

staining and 1.6% more ciliated cells with cilia staining compared to insufficient samples.

However insufficient samples had 23.1% more ciliated cells with even cytoplasmic staining

pattern. The Chi-squared statistical test gave a value of 66.8 with 13 degrees of freedom.

The P value was >0.0001 showing the data is extremely statistically significant and that it is

unlikely the data was as a result of chance.

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Comparing the proportion of ciliated epithelial cells in insufficient samples and

specimens with known secondary defects at TEM

Table 3 – Table showing the proportion of insufficient samples, Insufficient samples with

subsequent count, specimens with secondary defects and PCD patients at TEM with ciliated

epithelial cells with cilia

A total of 2519 ciliated cells were analysed at TEM. The insufficient sample group had a

large standard deviation of 26.5 for the total number of ciliated cells with cilia, showing the

data set was further away from the mean on average. There was a large difference between

the mean and median for each data set therefore non- parametric Mann Whitney U statistical

test gave a high T value of 12.5 and a large p-value of 5 which shows the differences in the

data set are not significant as the P value is larger than 0.05. The insufficient with a

subsequent count group had a standard deviation of 9.62. As there was large difference

between the mean and median the non-parametric Man Whitney U test gave a t-value of 33

and a large p-value of 21. Thus this data set is not significant at p< 0.05. The Secondary

defects group had a standard deviation of 29.4. Since the mean and median were similar

demonstrating normal distribution an independent T-test was performed which gave a t-value

of 0.8 and a p-value of 0.21 which is not significant at <0.05. The PCD group had a standard

deviation of 143.7. As there was a large difference between the mean and median the non-

parametric Mann Whitney U test gave a t-value of 14 and a p-value of 5 which is not

significant at p=<0.05. Thus the variance in these results was likely due to chance.

Insufficient Insufficient with count

Secondary defects PCD

Total number of ciliated cells counted 261 289 905 1064

Average ciliated cells counted per sample 29 32.1 150.8 29

Average ciliated cells with cilia counted per sample 22 24.3 137.6 25

Average % of ciliated cells with cilia 70.7 70.2 90.9 92.7

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The presence of intracytoplasmic cilia

No intracytoplasmic cilia were found during the analysis of ciliated cells at TEM. However

intracytoplasmic cilia had been found previously in some of the same samples, possibly from

other specimen grids.

Figure 7 - The EM micrograph on the left shows intracytoplasmic cilia from a patient with

confirmed PCD and the micrograph on the right show intracytoplasmic cilia from a patient

with secondary ciliary defects

Centrioles/ basal bodies within the cytoplasm

Centrioles and basal bodies localised in the cytoplasm rather than the apical surface of the

cell were seen throughout analysis and therefore this finding is not specific to defects of

centriolar docking.

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Figure 8: A photo micrograph from transmission electron microscopy showing the presence

of mislocalised basal bodies (a) which are normally located at the apical surface of the cell.

This is from an insufficient patient sample and was taken during the counting of ciliated

epithelial cells.

(a)

(a)

(a)

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Discussion

Retrospective study and its potential use in the PCD diagnostic service to diagnose

RGMC cases

The retrospective study demonstrates that RGMC cases in this particular cohort represents a

rare phenomenon (<1% of patients). The current protocol of diagnostic tests can be used to

identify secondary loss of cilia by using cell culture, nasal nitric oxide and repeat nasal brush

biopsy but distinguishing true RGMC cases remains difficult. Regular audit and follow up of

cases where samples were insufficient for diagnostic purposes should be conducted. Data

from this study suggests slides should be prepared for IF and samples should be fixed for

TEM and analysed despite there being low numbers of cilia. Thus making sure potential

candidate’s specimens are fixed for future electron microscopic analysis. The CCNO and

MCIDAS could be two of many possible RGMC gene defects where it can be said we have

only scratched the surface.

CEP164 staining characteristics and its potential use in identifying possible RGMC

cases

The staining pattern for CEP164 has been described and quantified in patient samples with

normal ciliary function and those who had reduced cilia number. There is a significant

difference in the staining patterns between healthy and insufficient specimens at

immunofluorescent analysis. The ciliated epithelial cells in the healthy samples showed a

similar staining pattern with an increased presence of ‘nuclear dots’ and nuclear staining

which may show normal cellular processes of ciliogenesis. These results resemble findings

reported in the MCIDAS study which finds that multicillin localised to the nucleus in control

cells destined for multiciliated cell differentiation whereas its expression in Mutant CCNO

cells is absent or very weak .1 The healthy samples also showed a much higher incidence of

the ‘gradient’ staining pattern which again may indicate ciliogenesis occurring with the

CEP164 protein involved in the generative process as mother centrioles move towards the

apical surface of the cell. Insufficient samples had nearly a quarter more ciliated cells with

even cytoplasmic staining which mirrored the findings in healthy samples showing that these

samples may have possible genetic mutations or secondary defects indicating defective

ciliogenesis and thus more uniform and insignificant pattern of CEP164 staining. This agrees

with the CCNO study which finds that CEP164 positive centrioles can be seen in mutant cells

likely caused by failure of correct centriole migration.12 A 1.6% difference in cilia staining

between the groups of samples showed that this is just an artefact from staining and didn’t

relate to the ciliogenesis status of the cells. The subsequent hypothesis testing for the data

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set showed that the differences between the normal and insufficient samples were significant

and unlikely to be caused by chance. Thus staining of CEP164 between these groups

showed substantial differences in staining patterns and can give an indication to the health

and ciliogenesis status. Normal specimens showed staining characteristics of well

differentiated epithelial cells. However IF using CEP164 could not be used to show potential

RGMC cases due to the difficulty in distinguishing defective ciliogenesis to secondary

defects.

Electron microscopy analysis and the presence of intracytoplasmic cilia

From the cell counts at electron microscopy the insufficient and insufficient with subsequent

count groups showed a lower average of ciliated cells with cilia at 70.7 and 70.2%

respectively, compared to specimens with secondary defects at 90.0% and PCD specimens

at 92.7%. This difference between the groups indicated possible RGMC cases in insufficient

samples from LM as well as differentiating insufficient samples with a substantial reduction in

cilia compared to samples with secondary defects. This agrees with findings from the CCNO

study where EM shows a reduced number of cilia in mutant airway cells, with 1 or 2 cilia per

cell.12 Statistical analysis shows that the differences within these groups are not significant

and are likely to be caused by chance. However this may be due to the small dataset

therefore more specimens included in the counts could then show a significant difference.

The appearance of mislocalised basal bodies was numerous throughout all samples,

especially in insufficient samples with ciliated cells with no cilia, similar to the MCIDAS study

where apical cell regions show normal microvilli composition and basal body mislocalisation

in the cytoplasm of patients with the gene defect.1 If the electron microscopy analysis was to

be repeated mislocalised basal bodies could be counted as this may give another aspect to

compare against to help further distinguish nude ciliated epithelial cells to secondary defects.

However distinguishing possible RGMC cases from patients affected by secondary defects

may be difficult as individuals with MCIDAS mutations presented with recurrent infections of

the upper and lower airways.1 In the CCNO study basal bodies and attached rootlets

mislocalised to the cytoplasm were found in mutant cells suggesting a basal body migration

defect.12 This feature could also be exploited as a tool to help reduce the number of patients

undergoing repeated brushing, whereby a change in practice could be implemented where

possible cilia RGMC could be sent for intracytoplasmic analysis.

In conclusion the hypothesis was rejected as RGMC does not appear to occur commonly in

patients referred for diagnosis of PCD. Review of repeated nasal brushings, staining and

electron microscopy analysis of number of cilia and centriole docking revealed no cases of

23

RGMC. Normal values have now been established for these tests in healthy and unhealthy

samples against which future patients with possible RGMC could be tested.

Future work

Antibodies to alternative downstream markers, such as FOXJ1, could be compared to

CEP164 antibodies, to show cilia generation and to help distinguish potential RGMC cases

from specimens affected by secondary influences. Mislocalised centrioles/ basal bodies

within the cytoplasm should be quantified at TEM to determine a normal range in healthy

patients with findings compared to the published research. This could ultimately be added as

an additional part of the diagnostic criteria for suspected cases of RGMC disorder.

24

Acknowledgements

Thank you to all the affected patients and families for their participation in the study. I am

grateful for the help of the Primary Ciliary Dyskinesia Diagnostic team in the Respiratory

Paediatrics unit and the Electron Microscopy unit at The Royal Brompton Hospital in London

for use of their transmission electron microscope. Especially Amelia Shoemark and Mellisa

Dixon who processed and found specimens for the study and helped with the completion of

the retrospective study using the department’s patient database. Thank you to the National

Heart and Lung Institute at the Emmanuel Kaye Building London for use of their fluorescent

microscope. Also to Imperial College London for use of their confocal microscope.

25

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