Thematic Review Series 2007
Respiration 2007;74:252–263
DOI: 10.1159/000101783
Genetic Causes of Bronchiectasis:
Primary Ciliary Dyskinesia
Hilda N. Morillas Maimoona Zariwala Michael R. Knowles
University of North Carolina, Chapel Hill, N.C., USA
Key Words
Bronchiectasis Mucociliary clearance Primary ciliary
dyskinesia DNAH5 DNAI1 Situs inversus Kartagener’s
syndrome
Abstract
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder reflecting abnormalities in the structure and
function of motile cilia and flagella, causing impairment of
mucociliary clearance, left-right body asymmetry, and sperm
motility. Clinical manifestations include respiratory distress
in term neonates, recurrent otosinopulmonary infections,
bronchiectasis, situs inversus and/or heterotaxy, and male
infertility. Genetic discoveries are emerging from familybased linkage studies and from testing candidate genes.
Mutations in 2 genes, DNAI1 and DNAH5, frequently cause
PCD as an autosomal recessive disorder. A clinical genetic
test has been recently established for DNAI1 and DNAH5,
which involves sequencing 9 exons that harbor the most
common mutations. This approach will identify at least one
mutation in these 2 genes in 25% of PCD patients. If biallelic mutations are identified, the test is diagnostic. If only
one mutation is identified, the full gene may be sequenced
to search for a trans-allelic mutation. As more disease-causing gene mutations are identified, broader genetic screening panels will further identify patients with PCD. Ongoing
© 2007 S. Karger AG, Basel
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investigations are beginning to identify genetic mutations
in novel clinical phenotypes for PCD, such as congenital
heart disease and male infertility, and new associations are
being established between ‘ciliary’ genetic mutations and
clinical phenotypes.
Copyright © 2007 S. Karger AG, Basel
Introduction
Mucociliary clearance (MCC) is a key component of
pulmonary defense mechanisms, which enables the airways to protect the lungs from potentially harmful substances in the surrounding environment, such as infectious organisms, allergens, irritants and chemicals. MCC
involves the regulation of ion transport by airway epithelium, ciliary function, and mucus secretion [1]. Disruption of this intricate defense system leads to clinical manifestations of lung disease. Cystic fibrosis (CF) is one example of a recessive disorder in which genetic mutations
of CFTR (cystic fibrosis transmembrane conductance
Previous articles in this series: 1. Contopoulos-Ioannidis DG,
Kouri IN, Ioannidis JPA: Genetic predisposition to asthma and atopy. Respiration 2007;74:8–12. 2. Sztrymf B, Yaïci A, Girerd B, Humbert M: Genes and pulmonary arterial hypertension. Respiration
2007;74:123–132.
Hilda N. Morillas, MD
CF/Pulmonary Research and Treatment Center
University of North Carolina at Chapel Hill
7006 Thurston Bowles Building, CB# 7248, Chapel Hill, NC 27599-7248 (USA)
Tel. +1 919 966 6780, Fax +1 919 966 7524, E-Mail hmorilla@med.unc.edu
regulator) cause a derangement of ion transport and subsequent ineffective MCC and cough clearance. Primary
ciliary dyskinesia (PCD; MIM 242650) is a genetic disorder of motile cilia, and the clinical manifestations reflect
ciliary dysfunction in lungs, sinuses, middle ear, male
fertility and organ lateralization [2]. Approximately 1 in
30,000 individuals are estimated to have PCD [3], but this
could be underestimated because of challenges in establishing the diagnosis. It is a genetically heterogeneous
disorder; however, in the majority of cases it is inherited
as an autosomal recessive trait. Patients with PCD have
impaired MCC, but retain cough clearance as a defense
mechanism [4].
Genetics have recently emerged as a critical feature to
understand the molecular pathogenesis of PCD and to
facilitate the diagnosis and allow better definition of the
clinical phenotypes. This article is designed to provide
the most current information on the status of genetic discoveries in PCD, which currently stem from family-based
genetic linkage studies, and from testing candidate genes
derived from knowledge of ciliary structure/function.
Understanding the structure and function of cilia can
lead to identifying more candidate genes and allow better
correlation between genetic mutations and clinical phenotype.
Table 1. Clinical manifestations of PCD
Organ
Clinical manifestation
Lung
Respiratory distress in term neonates
Bronchitis/recurrent infections
Bronchiectasis
Ears
Otitis media
Hearing loss
Cholesteatoma (after tympanostomy)
Nares/sinus
Chronic sinusitis
Polyposis
Genitourinary tract
Male infertility
Organ laterality
Situs inversus totalis
Situs ambiguus (heterotaxy), including
(a) Polysplenia or asplenia
(b) Vascular anomalies
(c) Complex congenital heart disease
Central nervous system
Hydrocephalus (rare)
Genetic-based (‘primary’) dysfunction of motile cilia
and sperm-tail flagella explains the complex PCD phenotype [3]. Common clinical presentations of the disease
include respiratory distress in full-term neonates, situs
inversus, recurrent otosinopulmonary infections, bronchiectasis, and male infertility; however, these manifestations vary by age and among patients [5] (table 1). In the
lung, MCC is coordinated by motile cilia that line the upper and lower airways. These rod-like organelles extend
from the airway epithelial cell surface and move extracellular mucus by synchronized beating. Immotile or dyskinetic respiratory cilia cause defective MCC, and affected
individuals have recurrent respiratory infections that
lead to chronic infections and inflammation of the upper
and lower airways [6]. The disease typically progresses to
overt bronchiectasis during late childhood or early adulthood and can ultimately cause chronic respiratory failure
[5]. Additionally, intrabronchial calcium deposition with
associated lithoptysis has been observed in a subset of
older patients [7]. The majority (95%) of PCD patients has
a history of recurrent otitis media requiring repeated an-
tibiotic treatments, and sinusitis is a universal feature in
PCD, demonstrating that ciliary function in the eustachian tube and sinuses is also critical for protection of
these organs [5]. Almost all male PCD patients are infertile, due to sperm immobility or dysmotility. In an individual patient, ultrastructural defects of sperm flagella
usually resemble those seen in respiratory cilia, but not
always [3]. Ciliary dysfunction in the fallopian tubes may
contribute to subfertility in affected women [8], although
this is difficult to validate.
Dysfunction of the embryonic node monocilia is associated with the complete mirror image of the usual arrangement of thoracic and abdominal organs (situs inversus totalis) in approximately half of the PCD patients
due to randomization of left-right body asymmetry [9]
(fig. 1, 2). PCD with chronic sinus and airway infections,
coupled to situs inversus, is also referred to as Kartagener’s syndrome (MIM 244400) [10]. Some PCD patients
have neither situs solitus nor situs inversus totalis, whereby some internal organs are located in the usual position,
but others are in a mirrored position or in duplicate [11].
This abnormality of organ location is termed situs ambiguus or heterotaxy. A recent review of 337 PCD patients
revealed that at least 6.3% (n = 21) had situs ambiguus; 12
of those 21 patients with heterotaxy had cardiac and/or
vascular abnormalities, and most (n = 8) had complex
congenital heart disease [11]. Thus, cilia-related genes are
excellent candidate genes for heterotaxy and congenital
PCD and Genetic Mutations
Respiration 2007;74:252–263
Defective Cilia and the Clinical Phenotype of PCD
253
Fig. 1. Random left-right asymmetry seen in patients with PCD. Chest radiographs of monozygotic twins with
PCD and ultrastructural defects of the IDA [5]. a Dextrocardia and dextrogastria in a patient with situs inversus totalis. b Situs solitus. Arrow is pointing to gastric bubble.
heart disease. Patients with situs inversus or heterotaxic
anomalies, particularly those with unexplained respiratory symptoms, are at risk of having an underlying defect
in ciliary structure, function, and genetics, and should be
evaluated for PCD. Conversely, PCD patients should have
a cardiac evaluation, particularly those with situs inversus or heterotaxy.
Management of PCD
Fig. 2. High-resolution chest tomography. The patient is a 24-
year-old female with PCD. Her ciliary electron micrograph
showed absent ODA and IDA. She has bronchiectasis (B) and situs
inversus totalis as demonstrated by a bilobed right lung and the
aorta (A) lying along the right side of the thoracic cavity.
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Respiration 2007;74:252–263
There are currently no treatments to correct the ciliary
dysfunction of PCD and standardized treatment has not
been defined [12]. Clinicians typically follow the general
guidelines for treatment of bronchiectasis, which frequently employs approaches used in CF. Clinical management involves enhancing MCC with physiotherapy, postural drainage, exercise, and/or mechanical oscillatory
vest percussion. Although there are no specific research
data on the efficacy of hypertonic saline, it can be used
empirically to improve MCC. Cough suppressants should
be avoided because cough is the only intact mechanism
for MCC in these patients [12]. A key component in the
management is prevention or rapid treatment of respiraMorillas/Zariwala/Knowles
Fig. 3. Electron micrographs (EM) of respiratory cilia. a EM image of a cilium from nasal epithelium of an unaffected individual. Normal-appearing ODAs (black arrow), IDAs and central apparatus are seen. b EM image
of a cilium from nasal epithelium of a patient with PCD homozygous for 10815delT mutation in DNAH5. This
patient has absent IDAs and ODAs. c EM image of a cilium from nasal epithelium of a patient with PCD. This
patient has normal-appearing ODAs but absent IDAs.
tory tract infections. Acute infectious exacerbations
should be managed with appropriate oral, intravenous or
inhaled antibiotics directed by the sputum culture [5, 12].
Patients should receive routine immunizations for respiratory pathogens, avoid exposure to pathogens, and avoid
smoking and other irritants that may increase mucus production. Localized bronchiectasis has previously been
managed by lobectomy, but its benefit is limited and
should be undertaken only after the involvement of experts. Surgical interventions may be required for specific
complications of chronic suppurative otitis media and sinusitis such as tympanostomy, nasal polypectomy, and
surgical sinus drainage. Lung transplant may be an option
for patients with end-stage lung disease [12].
The diagnosis of PCD is often delayed, which contributes to poor outcomes [13]. Early diagnosis in childhood
may allow treatment to delay, and possibly prevent, the
occurrence of bronchiectasis. For example, in the term
newborn, neonatal respiratory distress syndrome and
tachypnea can be the first signs of PCD, but this has only
recently been highlighted [5, 14, 15]. Therefore, educating
clinicians of the typical and atypical clinical features, in
addition to molecular and genetic research, is extremely
important.
The current gold standard for the diagnosis of PCD is
determining the ciliary ultrastructure defect via trans-
mission electron microscopy (TEM) or clearly documenting ciliary dysfunction via high-speed video microscopy
using standardized methodologies [15]. Studies in humans have revealed a spectrum of ultrastructural defects
(fig. 3), but the most common defects are absence of the
outer dynein arms (ODA) and/or inner dynein arms
(IDA). The frequency of isolated ODA defects range between 29 and 43%, isolated IDA defects range between 10
and 29%, and ODA plus IDA defects range between 7 and
57% [5, 16–18]. Other reported defects include absence of
the radial spokes, transposition (a peripheral microtubule
pair migrates to the center of the cilia because the central
pair is missing), and loss of central microtubules [5, 19].
As many as 15% of currently diagnosed PCD patients have
no ultrastructural defects [5, 16, 18, 20], but this number
is likely to escalate as genetic testing becomes more available, and the diagnosis can be firmly established in the
absence of gross ultrastructural abnormalities.
A major challenge for the diagnosis of PCD is that
TEM does not easily demonstrate all ‘primary’ (i.e., genetic) ultrastructural defects. For example, IDA defects
are difficult to define on TEM because of the low contrast
and the complexity of the composition and distribution
of the IDA. Carlen and Stenram [21] described that the
mean number of IDA per cilium was ! 0.6 for subjects
with PCD and 3–5 for control subjects, whereas the mean
number of ODA per cilium was !1.6 for subjects with
PCD, but 7.5–9.0 in control subjects. Computer-assisted
analysis of TEM cross-section photographs can sometimes improve IDA visualization [20].
PCD and Genetic Mutations
Respiration 2007;74:252–263
Diagnosis of PCD
255
From a genetic standpoint, different mutations are associated with specific ciliary ultrastructural phenotypes.
In cases in which no ultrastructural defects are identified, the diagnosis of PCD can be established only if the
patient has a compatible clinical phenotype, including situs inversus or heterotaxic defects, together with assessments of ciliary beat frequency and waveform, measurements of nasal nitric oxide (nNO) levels, or the use of
immunohistochemical immunofluorescence studies (see
details below) [6, 15]. Some patients with PCD may have
normal appearing ciliary activity, if viewed only with
light microscopy.
Acquired, or ‘secondary’, defects can occur with infection, allergic inflammation or environmental exposures,
and must be distinguished from primary genetic defects.
There are multiple changes in ciliary ultrastructure that
have been described as secondary changes, but loss of
ODA and/or IDA has not been described [22]. To aid in
distinguishing between primary and secondary ultrastructure abnormalities, respiratory epithelial cells can
be cultured, but only a few centers perform these techniques and a significant amount of cell material is necessary [18].
The current gold standard for determining ciliary dysfunction is high-speed video microscopy. Ciliated respiratory epithelium should be obtained via a brushing or
scraping of the inferior nasal turbinate. Strips of epithelium should be directly examined for ciliary beat frequency and pattern within 2 hours of obtaining the sample at centers familiar with the technique. There are several different methods to measure ciliary beat frequency
and to characterize the waveform of ciliary activity, but
there are no published data to support the superiority of
one method. The normal range is approximately 11–16
Hz, but this varies by temperature and other methodological variables, and experience is required in scoring
ciliary activity. It is important to remember that the patient should not have a respiratory infection at the time
of sampling because acute infections can cause secondary
changes in the function of cilia [15].
The production of nNO is reduced in patients with
PCD as demonstrated by Noone et al. [5] and Lundberg
et al. [23]. The test is performed by inserting an NO sampling line into one nostril and having the patient inhale
deeply and then close the soft palate. Although the mechanism in which nNO is reduced in patients with PCD is
still unclear, this test can clearly be used as a screening
tool, because there is a significant difference between the
nNO level in patients with PCD compared to normal and
disease controls [5]. However, it is key to rule out CF,
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since nNO levels in CF patients can overlap with levels of
PCD patients. Additionally, it is not invasive, simple to
perform, and compared to the saccharin test can be performed on small children.
The saccharin test was previously used as an inexpensive and simple procedure to screen adults and older children. A 1- to 2-mm piece of saccharin is placed 1 cm from
the anterior tip of the inferior nasal turbinate. The patient sits quietly with his or her head bent forward and
refrains from sniffing, coughing or eating during the
test. The time it takes for the patient to taste the saccharin is measured. An abnormal test is any length of time
greater than 60 min. A normal saccharin test clearly
demonstrates normal MCC but the test is time consuming and can be difficult to perform in patients unable to
cooperate [15]. This test is very nonspecific and now that
more definitive tests, such as TEM and videomicroscopy,
and genetic test are available, the saccharin test has no
clinical value.
Structure of Normal Cilia
Cilia are projections from the cell surface that are
bound by the cell membrane, and derived from the centriole. The ciliary axoneme is the cytoskeleton of the cilium composed of microtubules. Mammalian ciliary axonemes are formed with 2 major patterns. One pattern,
9 + 2, has nine doublet microtubules surrounding a central pair of singlet microtubules. Cilia containing this axonemal structure are usually motile and several hundred
of these cilia are located on airway epithelial cells. Major
structures that attach to the microtubules are the ODA
and IDA, which are the force-producing units of cilia and
flagella. There are approximately 4,000 dynein arms in
pairs per cilium. Other defined protein complexes are the
radial spokes, which radiate from the central pair, and the
nexin links, which connect the outer doublets [24, 25].
The other pattern is 9 + 0, in which the central pair is absent. These cilia are usually solitary and the molecular
motors, axonemal dyneins, which are responsible for ciliary movement, are also absent; therefore, these cilia are
nonmotile [26]. One exception is the embryonic node cilium, which is a motile, specialized monocilium with a
9 + 0 pattern with dynein arms [9]. It moves in a vortical
pattern distinct from the bending movement of the
9 + 2 cilia and plays a key role during embryogenesis for
determination of left-right asymmetry, which is thought
to result from the movement of liquid surrounding the
nodal cilium in a leftward direction [9, 27].
Morillas/Zariwala/Knowles
Ultrastructure of ODAs
To further understand the normal structure and function of human motile cilia, axonemal genes and proteins
of Chlamydomonas reinhardtii, a unicellular green alga
with two flagella that extend from its anterior end, have
been studied. Axonemal genes are highly conserved
throughout evolution [28], and human orthologs have
been identified for most genes that encode for axonemal
proteins. The ultrastructure of the Chlamydomonas flagellum has a distinctive ODA complex containing 3 heavy
chains (HCs), 2 intermediate chains, 9 light chains, and
3 docking complex proteins. The three HCs are , and
. Three human orthologs have been identified for the
Chlamydomonas HC, DNAH9 (chromosome 17p12),
DNAH11 (chromosome 7p21), and DNAH17 (chromosome 17q25). Two human orthologs have been identified
for the Chlamydomonas HC, DNAH5 (chromosome
5p15) and DNAH8 (chromosome 6p21) [29]. There have
been no human orthologs identified for the Chlamydomonas HC. This is consistent with the concept that the
ODA of vertebrates only have 2 HCs.
Because multiple orthologs have been identified, it is
thought that humans might have several types of ODA
complexes, and recent data support that notion. Fliegauf
et al. [30] used specific antibodies against DNAH5 and
DNAH9 and confocal immunofluorescence to localize
the ODA HC complexes along the axoneme. In wildtype respiratory cells, DNAH5 is present throughout the
entire length of the ciliary axoneme, whereas DNAH9
localizes only to the distal portion of the cilium. In contrast, in sperm cells the DNAH9 protein is present along
the entire length of the sperm tail and DNAH5 localizes
solely to the proximal part of the flagellum. Thus, there
are at least 2 types of ODA present in respiratory cilia
and sperm flagella. Type 1 stains positive for DNAH5
and negative for DNAH9, and type 2 ODA stains positive for both DNAH5 and DNAH9. This difference in
localization of different HCs in ODA complexes along
the longitudinal axis likely contributes to the characteristic beating pattern of respiratory cilia and sperm flagella [30].
patterns along the doublet microtubule of the flagellar
axoneme. The IDA has several subunits of heavy, intermediate, and light chains that are unique from the composition of ODA subunits, indicating different functions for these proteins [31]. Numerous human IDA HC
genes have been identified (DNAH1, DNAH2, DNAH3,
DNAH6, DNAH7, DNAH10, DNAH12, and DNAH14).
DNAH10 and DNAH2 are human orthologs of the
Chlamydomonas genes for the IDA HC1 and IDA
HC1, respectively. The othologs for the other 6 IDA HC
genes have yet to be identified. In addition, IDA intermediate-chain genes and IDA light-chain genes have
been identified and additional ortholog analyses are
ongoing [29].
Mutations in DNAI1
The ultrastructure and function of the IDAs are diverse and their arrangement along the axoneme is complex. Chlamydomonas exhibits seven different IDA isoforms which are organized with distinct localization
Genome-wide linkage analyses have determined that
there are likely multiple loci responsible for the mutations
that cause the PCD phenotype, which is compatible with
the observation that there are at least 250 proteins that
make up the ciliary axoneme [32]. Pennarun et al. [33]
confirmed the hypothesis that mutations in human genes
homologous to Chlamydomonas genes are responsible for
PCD by demonstrating that mutations in DNAI1 are
present in patients with PCD lacking ciliary ODAs. A
mutation in IC78, the Chlamydomonas homolog of
DNAI1, results in the inability to assemble the ODA and
decreased motility of Chlamydomonas [34]. DNAI1 was
the first human gene identified in which mutations cause
known ultrastructural defects described in PCD patients.
DNAI1, located at chromosome 9p13-21, is expressed in
the tracheobronchial tree and testes and is composed of
20 exons [33].
In the initial study, two loss-of-function mutations of
DNAI1 (IVS1+2_3insT and 282_283insAATA) were
identified in a patient with immotile cilia and absent
ODA [33]. Subsequent studies of 226 unrelated PCD patients identified 18 different mutations in DNAI1, ranging from nonsense mutations to splice and missense mutations [35–37] (table 2). A high prevalence (55%) of the
mutant alleles had the IVS1+2_3insT mutation, which is
known to be a founder mutation. The most common mutant alleles were found to cluster in intron 1 and exons 13,
16 and 17. Approximately 50% of unrelated patients carried at least one mutant allele in these 3 exons. In addition, DNAI1 mutations were present in 11% of 1200 unrelated Caucasian patients tested and in 14% of patients
with ODA defects. It is notable that none of the patients
PCD and Genetic Mutations
Respiration 2007;74:252–263
Ultrastructure of IDAs
257
Table 2. DNAI1 mutations in patients
with the classic clinical phenotype for
PCD
Type of mutation
Nucleotide mutation
Protein change
Exon/intron
Nonsense or
frameshift
282_283insAATA
463delA
874C>T
1212T>G
1307G>A
1644G>A
1657_1668del
1704G>A
1926_1927insCC
G95NfsX24
T155LfsX18
Q292X
Y404X
W436X
W548X
T553_F556del
W568X
I643PfsX48
exon 5
exon 6
exon 10
exon 13
exon 13
exon 17
exon 17
exon 17
exon 19
Splice
IVS1+2_3insTa–c
IVS7–2A>G
IVS10–4_7delGTTT
1490G>Ac
IVS19+1G>Ac
truncation of protein
splice mutation
splice mutation
R468_K523del
A607_K667del
intron 1
intron 7
intron 10
exon 16
intron 19
Missense
1222G>A
1543G>Ab
1612G>Ab
1703G>C
V408M
G515S
A538T
W568S
exon 13
exon 16
exon 17
exon 17
Genetic material from 226 unrelated patients has been analyzed for DNAI1 mutations. Common mutations cluster in intron 1 and exons 13, 16 and 17. 55% of the mutant
alleles had the IVS1+2_3insT mutation. Identification of these common mutations has
led to the development of a diagnostic genetic test panel (see text for details). Mutations
within the DNAI1 gene are always associated with ODA defects [37].
a
Founder mutation; leads to truncation of protein.
b Found in 2 or more unrelated patients.
c RNA tested.
with IDA defects had mutations in DNAI1 [37]. Thus, the
founder mutation, IVS1+2_3insT, and the exon cluster of
mutations, are useful genetic markers for PCD.
Mutations in DNAH5
Mutations in the Chlamydomonas HC of the ODA
cause a slow swimming Chlamydomonas with ultrastructural abnormalities in the ODA [38]. The human HC ortholog, DNAH5 (chromosome 5p15), was identified as a
gene responsible for causing PCD by homozygosity mapping in a large consanguineous Arabic family, where affected individuals lacked ODAs by TEM. The DNAH5
genomic region spans 250 kb and is made up of 79 exons
and 1 alternative exon [39]. Sequencing the 80 exons in a
total of 134 unrelated patients with PCD for the presence
of DNAH5 mutations revealed a large number of mutations [40]. Mutations in DNAH5 were identified as disease-causing in 28% of all unrelated patients, and present
in over half (53%) of the unrelated patients with ODA de258
Respiration 2007;74:252–263
fects. None of the individuals with IDA ultrastructural
defects had mutations in DNAH5. Therefore, DNAH5
mutations are a major cause of ODA ultrastructural defects. It is noteworthy that approximately 75% of patients
had biallelic mutations in DNAH5, whereas approximately 25% had only one mutation, despite complete sequencing of the coding region [40]. We speculate that many of
these patients will have small exonic deletions, as seen in
other recessive disorders, such as CF [41], and studies to
test this hypothesis are underway.
There are multiple types of mutations in DNAH5, including nonsense or frameshift mutations, missense mutations, and splicing mutations [40] (table 3). Six mutations (10815delT; 5563_5564insA; 13458_13459insT;
IVS76+5G1A; 8528T1C, and 8440_8447delGAACCAAA) were found in more than one unrelated families.
Three of the mutations were related to ancestral founders: 10815delT; IVS76+5G1A, and 8528T 1C. It was also
noted that the 10815delT mutation was more common in
a population of USA patients, as compared to patients
from Germany [40].
Morillas/Zariwala/Knowles
Table 3. DNAH5 mutations in patients
with the classic clinical phenotype for
PCD
Type of mutation Nucleotide mutation
Protein change
Exon/intron
Nonsense
or frameshift
232C>T
252T>G
832delG
1426_1427delTT
1627C>T
1828C>T
3905delT
4360C>T
5130_5131insA
5281C>T
5482C>T
5563_5564insAb
5599_5600insC
6037C>T
7914_7915insA
7915C>T
8440_8447delGAACCAAAb
8029C>T
8167C>T
8314C>T
8404C>T
8910_8911delATinsG
10815delTa, b
13194_13197delCAGA
13426C>T
13458_13459insTb
13486C>T
R78X
Y84X
A278RfsX27
F476SfsX26
Q543X
Q610X
L1302RfsX19
R1454X
R1711TfsX36
R1761X
Q1828X
I1855NfsX5
I1867PfsX35
R2013X
R2639TfsX19
R2639X
E2814fsX1
R2677X
Q2723X
R2772X
Q2802X
2970SfsX7
P3606GfsX23
D4398EfsX16
R4476X
N4487fsX1
R4496X
exon 3
exon 3
exon 7
exon 11
exon 12
exon 14
exon 25
exon 28
exon 32
exon 33
exon 33
exon 34
exon 34
exon 36
exon 48
exon 48
exon 50
exon 49
exon 49
exon 50
exon 50
exon 53
exon 63
exon 76
exon 77
exon 77
exon 77
Splice
1730G>Cc
IVS17+2T>C
IVS27+1G>A
IVS74–1G>Cc
IVS75–2A>T
IVS76+5G>Aa, b
N549_R577delfsX5
splice mutation
splice mutation
S4304DfsX6
splice mutation
splice mutation
exon 13
intron 17
intron 27
intron 74
intron 75
intron 76
Missense
4361G>A
5147G>T
6791G>A
7039G>A
7502G>C
8528T>Ca, b
10226G>C
10555G>C
11528C>T
12614G>T
R1454Q
R1716L
S2264N
E2347K
R2501P
F2843S
W3409S
G3519R
S3843L
G4205V
exon 28
exon 32
exon 41
exon 43
exon 45
exon 51
exon 60
exon 62
exon 67
exon 73
The most common mutations cluster within exons 34, 50, 63, 76 and 77. These 5 exons
contain >50% of the DNAH5 mutations seen in 134 unrelated patients that were analyzed. Identification of these common mutations has led to the development of a diagnostic genetic test panel (see text for details) [40].
a Founder mutation.
b Found in 2 or more unrelated patients.
c RNA tested.
PCD and Genetic Mutations
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259
Electron-microscopic photographs of respiratory cilia
from three unrelated patients with homozygous mutations of DNAH5 were compared to correlate genotype
and phenotype. Mutations causing premature translational termination of DNAH5 (I1855NfsX5 and
E2814fsX1) resulted in a complete absence of all ODA in
respiratory cilia. In contrast, a splice site mutation (IVS74–
1G1C) did not cause total absence of ODA, but rather an
ODA ‘stub’, indicating a partial ODA structural deficiency with loss of function [42].
Testing for Mutations in DNAI1 and DNAH5
The extensive research on the genetic mutations causing PCD has led to the development of a genetic test. Utilizing the information of founder mutations and the clustering of mutations in certain exons, selective mutational
analysis of DNAI1and DNAH5 can reduce the labor-intense process of sequencing entire genes searching for
mutations. By sequencing only 9 (out of 100) exons and
the adjacent splice sequences that harbor the most common mutations in DNAI1 or DNAH5, at least one mutant
allele can be detected in approximately 38% of PCD patients [37]. This test becomes diagnostic if biallelic mutations are identified. If only one mutation is identified, the
full gene will be sequenced to search for a trans-allelic
mutation [37]. Identification of other additional diseasecausing genes will facilitate the diagnosis in PCD, including patients with a nonclassic clinical phenotype, and
help to screen other patient populations at risk.
Other Genes with Disease-Causing Mutations
Other mutations that cause PCD are found in 3 genes:
DNAH11, RPGR and OFD1. DNAH11 is located on chromosome 7, along with CFTR, and encodes for an axonemal ODA HC. There was 1 patient with a homozygous
nonsense mutation in DNAH11 and a homozygous
F508 mutation in CFTR, reflecting uniparental isodisomy, in which both chromosome pairs were inherited
from only one parent. This patient had situs inversus and
severe respiratory phenotype, but normal ciliary ultrastructure [43]. The iv/iv mouse has a homozygous missense mutation in the mouse ortholog (Dnah11; lrd) that
causes the embryonic node cilia to be immotile. Affected
mice have random left-right axis asymmetry, but lack respiratory symptoms. It is still unknown whether DNAH11
mutations are a cause of PCD or only cause random left260
Respiration 2007;74:252–263
right asymmetry [44]. There are ongoing studies to further evaluate the possibility that mutations in DNAH11
can cause the full clinical phenotype of PCD.
Retinitis pigmentosa (RP) is a rare heterogeneous genetic disorder that affects the photoreceptors on the retinal epithelium and leads to vision loss. RPGR (RP guanosine triphosphate regulator) facilitates the transport of
proteins across the connecting cilium of rod and cone
photoreceptors and is expressed in bronchial and sinus
epithelial cells [45, 46]. In one study, 2 siblings with RP
and symptoms of PCD, such as otosinopulmonary disease, had abnormal ciliary function and structure. Both
patients had some cilia with normal-appearing structures and other cilia missing IDAs and/or ODAs. A deletion and a missense mutation in RPGR were identified in
both patients, while molecular studies in 13 other unrelated families with PCD did not reveal any mutations in
RPGR [47].
Oral-facial-digital type 1 (OFD1) syndrome is an Xlinked dominant developmental disorder characterized
by craniofacial malformations, digital abnormalities and
mental retardation, caused by mutations in OFD1 and is
usually lethal in males [48]. OFD1 is localized at the centrosome and basal body of the primary cilia, and is required for the formation of primary cilia. Mice with heterozygous Ofd1 mutations have several phenotypes, including embryonic lethality, the absence of cilia in the
embryonic node, and midline defects of the cardiac tube
[49]. A large Polish family with a novel X-linked recessive
mental retardation syndrome and ciliary dysfunction
had 9 affected males with developmental delay and a clinical phenotype compatible with PCD, including chronic
respiratory problems and full-term neonatal respiratory
distress. PCD was diagnosed by high-speed video microscopy, which revealed dyskinetic cilia with a disorganized beating pattern. Genomic analysis of the affected
members revealed a frameshift mutation in the OFD1
gene, and is the only example thus far linking OFD1 mutations with the clinical features of PCD [50].
Candidate Genes That Have Tested Negative for PCD
Human orthologs of Chlamydomonas genes known to
cause ultrastructural and functional defects are potential
disease-causing genes in patients with similar axonemal
defects. Multiple candidate genes have been tested in
PCD patients and their families, but no other diseasecausing mutations have been identified (table 4).
Morillas/Zariwala/Knowles
Table 4. Candidate genes without disease-causing mutations for PCD
Ciliary ultrastructure
Chlamydomonas
protein
Human gene
Number of PCD
families tested
ODA HC
HC
HC
HC
none identified
DNAH9
DNAH17
ODA IC
IC2 (IC69)
DNAI2
16
Bartoloni et al., 2000 [53]; Pennarun et al., 2002 [54]
ODA LC
LC1
LC6
LC2
LC8
DNAL1
DNAL4
TCTE3
LC8
86
54
36
58
Horvath et al., 2005 [55]
Gehrig et al., 2002 [56]
Neesen et al., 2002 [57]
Bartoloni et al., 2000 [53]
IDA HC
unknown
unknown
DNAH3
DNAH7
7
1
IDA IC
IC140
IC140
126
IDA LC
P28
HP28
61
Gehrig et al., 2002 [56]; Pennarun et al., 2001 [61]
IDA, other
none
DPCD
51
Zariwala et al., 2004 [62]
2
4
References
Bartoloni et al., 2001 [51]
Blouin et al., 2003 [52]
Blouin et al., 2002 [58]
Zhang et al., 2002 [59]
Knowles et al., 2006 [60]
IC = Intermediate chain; LC = light chain [24].
Conclusion
PCD is a genetically heterogeneous disorder that
causes abnormalities in the structure and function of cilia and leads to recurrent otosinopulmonary infections,
bronchiectasis and is associated with situs inversus and
heterotaxy. Two genes, DNAI1 and DNAH5, have been
identified to have many disease-causing mutations, and
some of these occur in multiple patients. Based on these
data, a clinical genetic test is available at the University of
North Carolina at Chapel Hill (USA) and the University
Hospital Freiburg (Germany). This screening test sequences the 9 exons and adjacent sequences that harbor
the most common gene mutations of DNAI1 and DNAH5
and can identify the causative mutation on at least one
allele in 25% of all PCD tested. Additional, novel diseasecausing genes will likely be discovered by investigating
other candidate genes, and by genome-wide linkage-
References
PCD and Genetic Mutations
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disease-causing gene mutations are identified, they will
be added to genetic screening panels to further identify
patients with PCD. In addition, investigations to link genetic mutations to clinical phenotypes, such as in patients
with congenital heart disease and male infertility, will aid
in identifying new associations between genetic mutations and clinical phenotypes.
Acknowledgments
We thank Peader G. Noone, MD for critically reading the
manuscript. We also gratefully acknowledge the UNC clinicians,
researchers and collaborators for providing care to PCD patients
and continually searching for more information, especially Dr.
Margaret Leigh.
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