1. 1
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: ……………………

2. 2
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.

3. 3
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-

4. 4
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

5. 5
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,

6. 6
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).

7. 7
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

8. 8
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.

9. 9
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.

10. 10
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

11. 11
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).

12. 12
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.

13. 13
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.

14. 14
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)

15. 15
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.

16. 16
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
2 1 2 Not performed Too young for
nasal NO
3 3 2 Not performed Too young for
nasal NO
4 1 1 Not performed Too young for
nasal NO
5 1 0 Not performed 412
6 2 0 Not performed Too young for
nasal NO
7 1 1 Not performed Too young for
nasal NO
8 1 1 Not performed Too young for
nasal NO
9 1 1 Not performed 9
10 1 1 Not performed Too young for
nasal NO
11 1 0 Not performed 7.02

17. 17
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.

18. 18
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

19. 19
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.

20. 20
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)

21. 21
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

22. 22
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. 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. 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. 25
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