Static respiratory cilia associated with mutations in Dnahc11/DNAH11: A mouse model of PCD


  • Jane S. Lucas,

    1. Primary Ciliary Dyskinesia Group, Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, UK
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  • Elizabeth C. Adam,

    1. Primary Ciliary Dyskinesia Group, Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, UK
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  • Patricia M. Goggin,

    1. Primary Ciliary Dyskinesia Group, Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, UK
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  • Claire L. Jackson,

    1. Primary Ciliary Dyskinesia Group, Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, UK
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  • Nicola Powles-Glover,

    1. MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK
    2. Current address AstraZeneca R&D, Alderley Park, Macclesfield, UK
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  • Saloni H Patel,

    1. MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK
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  • James Humphreys,

    1. MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK
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  • Martin D. Fray,

    1. MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK
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  • Emilie Falconnet,

    1. Department of Genetics and Development, University of Geneva, Geneva, Switzerland
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  • Jean-Louis Blouin,

    1. Department of Genetics and Development, University of Geneva, Geneva, Switzerland
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  • Michael T. Cheeseman,

    1. MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK
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  • Lucia Bartoloni,

    1. Department of Genetics and Development, University of Geneva, Geneva, Switzerland
    2. Department of Clinical Pathology, ULSS12 Veneziana, Venezia, Italy
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  • Dominic P. Norris,

    Corresponding author
    1. MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK
    • MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK
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  • Peter M. Lackie

    Corresponding author
    1. Primary Ciliary Dyskinesia Group, Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, UK
    • Primary Ciliary Dyskinesia Group, Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton NIHR Respiratory Biomedical Research Unit, Southampton General Hospital, Southampton, SO16 6YD, UK
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  • Communicated by Ming Qi


Primary ciliary dyskinesia (PCD) is an inherited disorder causing significant upper and lower respiratory tract morbidity and impaired fertility. Half of PCD patients show abnormal situs. Human disease loci have been identified but a mouse model without additional deleterious defects is elusive. The inversus viscerum mouse, mutated at the outer arm dynein heavy chain 11 locus (Dnahc11) is a known model of heterotaxy. We demonstrated immotile tracheal cilia with normal ultrastructure and reduced sperm motility in the Dnahc11iv mouse. This is accompanied by gross rhinitis, sinusitis, and otitis media, all indicators of human PCD. Strikingly, age-related progression of the disease is evident. The Dnahc11iv mouse is robust, lacks secondary defects, and requires no intervention to precipitate the phenotype. Together these findings show the Dnahc11iv mouse to be an excellent model of many aspects of human PCD. Mutation of the homologous human locus has previously been associated with hyperkinetic tracheal cilia in PCD. Two PCD patients with normal ciliary ultrastructure, one with immotile and one with hyperkinetic cilia were found to carry DNAH11 mutations. Three novel DNAH11 mutations were detected indicating that this gene should be investigated in patients with normal ciliary ultrastructure and static, as well as hyperkinetic cilia. Hum Mutat 33:495–503, 2012. © 2011 Wiley Periodicals, Inc.


Cilia are hair-like membrane bound, microtubule-based structures seen in most cells in the body. Immotile primary cilia and motile cilia both contain a microtubule skeleton or axoneme, comprising nine microtubule doublets equally spaced around the periphery of the cilium. A central microtubule pair is also evident in motile cilia such as those in the respiratory tract. At least 250 proteins are associated with the ciliary axoneme [Dutcher, 1995; Gherman et al., 2006; Ostrowski et al., 2002]. Dynein arms on the peripheral microtubules are mechanochemical ATPases powering ciliary movement [Mitchison and Mitchison, 2010]. Motile cilia have multiple roles in human health and disease including respiratory mucociliary clearance and correct specification of left-right (L-R) patterning during embryonic development [Baker and Beales, 2009].

Static or abnormally motile cilia result in primary ciliary dyskinesia (PCD), a disease estimated to affect between 1 in 15,000–30,000 live births [Barbato et al., 2009]. It is caused by genetically heterogeneous, autosomal recessive mutations and results in defective mucociliary clearance [Bush et al., 2007]. PCD is characterized by chronic upper and lower respiratory tract infections with bronchiectasis becoming more prominent with age [Bush et al., 2007]. Male infertility can result from abnormal sperm motility, the sperm tail being structurally homologous with cilia. Early diagnosis of PCD has been highlighted as being clinically important to reduce both short- and long-term morbidity and allow genetic counseling [Bush et al., 2007]. Ultrastructural defects in the axoneme underlie a significant proportion of PCD cases, most frequently defects of dynein arms [Bush et al., 2007]. However, at least 10% of PCD patients have normal ciliary ultrastructure [Greenstone et al., 1983; Jorissen et al., 2000; Papon et al., 2010] and diagnosis often proves particularly challenging in these patients. Two mutations in the dynein heavy chain gene DNAH11 (MIM# 603339) were recently described in a family with PCD associated with hyperkinetic cilia but normal ultrastructure [Schwabe et al., 2008]. Four independent cases with different DNAH11 mutations have also been described [Bartoloni et al., 2002; Berg et al., 2011; Pifferi et al., 2010] although the overall prevalence of DNAH11 mutations in PCD is unknown.

Motile cilia in the embryonic node drive a leftwards “nodal flow”, directing asymmetric embryonic gene expression and ultimately morphology [Nonaka et al., 2002]. Inverted L-R asymmetry (situs inversus) has long been linked to PCD and is found in approximately 50% of patients [Kartagener and Horlacher, 1935]. This randomization is believed to result from dysfunctional nodal cilia leading to defective nodal flow [Nonaka et al., 2002]. The classical mouse mutant, inversus viscerum (Dnahc11iv; iv), was originally identified as demonstrating randomized situs [Hummel and Chapman, 1959] and was subsequently shown to be accompanied by immotile nodal cilia and absent nodal flow [Okada et al., 1999]. It results from a missense (E2271K) mutation in the AAA2 domain of Dnahc11, the mouse homolog of DNAH11 [Supp et al., 1997; Supp et al., 1999]. Subsequently, in a morphology-based study, tracheal cilia and sperm were reported to demonstrate normal motility [Handel and Kennedy, 1984]. Other alleles include an engineered null allele Dnahc11tm1Ssp [Supp et al., 1999] and a splicing mutant Dnahc11lrm3 (lrm3) that lacks 50 amino acids from the hinge region [Ermakov et al., 2009] both similarly demonstrate L-R defects, although nodal cilia motility has not been assayed in these alleles.

A number of potential mouse PCD models have been characterized. A significant proportion, including Dnahc5 (MIM# 603335) [Tan et al., 2007], Poll (MIM# 606343) [Kobayashi et al., 2002], and Dnaic1 (MIM# 604366) [Ostrowski et al., 2010], demonstrate additional defects, particularly hydrocephalus and cardiac anomalies, reducing their viability and raising significant animal welfare issues. The repeated demonstration that mutation of human PCD loci in the mouse results in hydrocephalus has led some to suggest an underlying difference in physiology [Ibanez-Tallon et al., 2002; Ostrowski et al., 2010]. The nm1054 mouse (MGI: Del(1)1Brk), a six-gene deletion, gives rise to PCD-like upper respiratory pathology, lacks cardiac defects, and only shows hydrocephalus on certain genetic backgrounds. These mice, however, show loss of sperm tails and lethal anemia. Transgenic rescue demonstrated Pcdp1 (MGI: Gm101) to underlie the PCD-like symptoms and Steap3 the lethal anemia [Lee et al., 2008; Ohgami et al., 2005], permitting genetic rescue of the anemia. The Dnahc1 (MIM# 603332) null results in reduced tracheal cilia and sperm tail motility; however, no analysis of upper respiratory tract pathology was performed making it unclear how well this models PCD. A preliminary analysis of Ttll1 (MIM# 608955) mutants [Vogel et al., 2010] demonstrates PCD-like upper respiratory pathology, but ciliary motility and morphology has yet to be studied. These data led Ostrowski and colleagues to engineer postnatal deletion of Dnaic1 (MIM# 604366); deletion at 8–12 weeks of age avoids hydrocephalus. This, however, requires additional experimental intervention and clearly cannot model the condition in juveniles, the stage at which the disease is first evident in humans [Ostrowski et al., 2010]. Whether an easy-to-use mouse model of the respiratory PCD phenotypes exists, lacking additional phenotypes, remains to be determined.

To investigate the potential of iv mice as a model of PCD, we examined ciliary function, ciliary ultrastructure, and pathological read-outs of PCD-related disease (including otitis media) that often accompany PCD in humans. Previous studies have suggested normal tracheal ciliary and sperm motility in iv mice on the basis of ultrastructure but did not include motility assays [Handel and Kennedy, 1984]. We found tracheal cilia to be static at 37°C, consistent with a PCD phenotype and pathological evidence of PCD-like symptoms in the upper respiratory tract. These data argue that iv models aspects of human PCD in the mouse. In parallel to our mouse model analysis, we reviewed the data of consecutive patients referred to our national PCD diagnostic service (2006–2009). Twenty-eight patients were diagnosed with “classical PCD” defined by abnormal ciliary function plus diagnostic ciliary transmission electron microscopy (TEM), and 241 patients had PCD excluded on the basis of light microscopy ± electron microscopy (“not PCD”). Two unrelated patients had a clinical phenotype strongly indicative of PCD but with normal ciliary ultrastructure. We tested these patients and both were found to carry novel mutations in DNAH11.



Mice were bred in a specific pathogen free (SPF) environment at MRC Harwell. Inbred Dnahc11iv (iv) mice are directly descended from the mixed background stock described by Hummel and Chapman [Hummel and Chapman, 1959]. Congenic iv/+ mice were derived by 10 generations of backcrosses to C3H/HeH (C3H); C3H-congenic experimental cohorts were from congenic iv/+ intercrosses. C3H/HeH congenic Dnahc11lrm3 (lrm3) are as previously described [Ermakov et al., 2009]. SNP genotyping was for the published mutations [Ermakov et al., 2009; Supp et al., 1997]. Samples were collected postmortem, following terminal anaesthesia using sodium pentobarbitone (Euthatal). Control and iv/iv samples were coded and researchers blinded to both genotype and phenotype (situs). The mouse age, gender, and cohort size is presented in Table 1. Fertility testing was conducted by placing two C57Bl/6J females each with six C3H/HeH congenic iv/iv males and six +/+ sibling control males. Copulation plugs were checked for daily over one week. At 8.5 d.p.c. plugged females were sacrificed, uterine horns dissected, and the number of implanted embryos counted. All males and females used were age matched.

Table 1. Mouse Genotype and Group Characteristics
  1. N/D, not done.

Genetic backgroundMixedMixedMixedMixedC3H/HeHC3H/HeHC3H/HeH
Females  5102214
Age (w) or range591531–4214–2313–2511
Situs inversus3/50/54/106/193/50/5N/D
Otitis media0/52/50/102/190/50/50/20
Lung histology0/5 no abnormalities0/5 no abnormalities3/10 peri-bronchiolar lymphoid aggregates1/19 peri-bronchiolar lymphoid aggregates1/5 peri-bronchiolar lymphoid aggregates1/5 peri-bronchiolar lymphoid aggregatesN/D


Sections of mouse lung, snout, and middle ear were produced following standard necropsy and histological protocols [Brown et al., 2005; EUMORPHIA Consortium, 2007]. Briefly, trachea and lungs were inflated with 10% neutral buffered formalin (NBF) then immersion fixed along with the head. Decalcification was as described in Parkinson et al. [Parkinson et al., 2006]. Samples were paraffin wax embedded, sectioned, and stained with haematoxylin and eosin according to standard protocols [Brown et al., 2005; EUMORPHIA Consortium, 2007].

Mouse Sperm Analysis

Five iv/iv C3H and five C3H male mice were sacrificed by cervical dislocation according to Schedule 1 of the UK Animals (Scientific Procedures) Act 1986. The vas deferens and cauda epididymus were dissected out and placed in synthetic human tubal fluid (HTF) under mineral oil (Sigma, Poole Dorset, UK). Motile spermatozoa were expressed from the vas deferens by lacerating the tissue with a 25-gauge hypodermic needle six times and then gently massaging the cauda epididymis with a pair of fine forceps. Samples were incubated for 1 hr at 37°C, 5% CO2, allowing the sperm to disperse fully, then diluted 1:10 in HTF and loaded onto preformed 20 µl four-chamber capillary slides (Leja, Nieuw-Vennep, The Netherlands). Analysis was with IVOS (Integrated Visual Optical System for sperm analysis) sperm analyzer (Hamilton-Thorne Biosciences, Beverly, MA) previously optimized for mouse sperm. Each sample was analyzed three times, and five fields counted per reading. Samples of epididymus were also prepared for electron microscopy.


The research was approved by the Southampton and South West Hampshire Local Ethics Committee (LREC No 07/Q1702/109). Two patients with a clinical history consistent with PCD including abnormal ciliary function, but with normal axoneme ultrastructure, consented to genetic analysis. Clinical details are summarized in Table 2. Comparative data are given for consecutive “classical” PCD patients with ultrastructural anomalies and patients referred to the centre for PCD diagnostics who had the diagnosis excluded.

Table 2. Clinical Details with Local Reference Values
 Patient #730Patient #616“Classical PCD” n = 28 (%)“Not PCD” n = 241 (%)
  1. The clinical characteristics of the index patients (#730 and #616), 28 consecutive patients seen in the Southampton PCD service diagnosed with “classical PCD” defined by abnormal ciliary function plus diagnostic ciliary TEM, and 241 consecutive patients (2006–2009) in whom PCD was excluded on the basis of light microscopy ± electron microscopy (“not PCD”).

  2. aHeterotaxy, left or right isomerism; complex cardiac disease.

  3. ODA ± IDA: outer dynein arm defects ± inner dynein arm defects.

  4. bNeonatal pneumonia, admission to neonatal unit or respiratory distress at birth. Data from 26 patients.

  5. cEight patients (3.3%) had a mean CBF below 11 Hz, but this was deemed a secondary defect on the basis of other results.

  6. dIf defects fall below 60%, then the biopsy is considered nonPCD according to local reference data.

Age at test/diagnosis (years)460–67.7 Mean 13.4; Median 5.30–67 Mean 16.4 Median 9.0 SD 19.4
Neonatal symptomsbYesYes14 (50%) 
Persistent cough Persistent, wet coughYesYes25 (89%) 23 (82%)207 (86%) 152 (63%)
Persistent/frequent rhinitisYesYes22 (79%)128 (53%)
Sinusitis  5 (18%)48 (20%)
Middle ear effusionsYesYes16 (57%)42 (17%)
Situs inversus totalisSitus solitusSitus solitus8 (29%)10 (4%)
Heterotaxya  3 (11%)6 (2%)
Nasal NO (ppb)4924n = 19 Range 2–70; Mean 33.7; SD 19.4n = 59 Range 80->2000 Mean 570 SD 350
Electron microscopyWithin normal limitsdWithin normal limitsd Within normal limitsd [n = 169]
ODA ± IDA  25 (89%) 
Radial spoke defect  3 (11%) 
Ciliary FunctionRapid, erratic, dyskinetic ciliary beating; CBF was immeasurable n = 2 assessmentsStatic cilia with occasional slow ciliary activity (mean CBF = 5.26 Hz SEM ± 0.63 by FFT analysis at one assessment) n = 3 assessments24 static; 1 twitching; 3 dyskinetic8.6–23.0c Mean 15.1; median 15.0 SD 2.5; all patterns within normal limits

Ciliary Beat Analysis

Mouse tracheal rings were suspended in MEM (Invitrogen, Paisley, UK). For each sample, ciliary beat frequency (CBF) was determined at 37 °C at six independent points. Fast Fourier transform (FFT) analysis of a digitized photodiode signal from an Olympus inverted microscope (Olympus UK, KeyMed, Southend on Sea, UK) identified the dominant signal frequency. Human nasal epithelial cells were obtained from the inferior turbinate using disposable 2-mm Olympus EndoTherapy BC-202D cytology brushes (Olympus UK). Cells were resuspended in 1.5 ml supplemented BEGM medium (CC3170 Clonetics, Lonza, Walkersville Inc., MD) at 37°C and 10 video microscopy sequences recorded at 250 frames per second (fps) using a FASTCAM high-speed video camera with a 100× oil immersion objective. CBF and beat pattern analysis were assessed by an experienced observer at 30 fps.

Electron Microscopy

Mouse and human samples were prepared following standard TEM and scanning electron microscopy (SEM) protocols [Bozzola and Russell, 1991; Rutland et al., 1982]. For each sample, 300 cilia cross- sections were examined at ×30,000 magnification; ultrastructural defects in dynein arms, microtubules or deviations from the normal 9+2 microtubule arrangement were counted by an expert observer.

Genetic Analysis

The 82 DNAH11 exons were amplified with intronic primers as previously described [Bartoloni et al., 2002] and directly sequenced. Sequence variants were named according to NM_003777.3 cDNA and NP_003768.2 protein sequences and compared with the latest release of the Single Nucleotide Polymorphism database (dbSNP build 131). Variants not in dbSNP were compared with sequences from 96 PCD patients; Polyphen software was used to predict their effect on the protein.


iv Tracheal Cilia are Immotile

Tracheal slices were dissected from inbred iv/iv and age-matched wild-type C3H mice (Fig. 1). Wild-type tracheal slices exhibited mean CBF of 18.7 Hz (SEM = 0.35 Hz, n = 6), comparable to previously published data [Lechtreck et al., 2008; Sisson et al., 2003]. In contrast, no cilia motility was detected in samples from iv/iv mice (n = 6). In light of these results and as the original homozygous iv colony is maintained on a unique inbred background, congenic C3H.iv mice were produced and analyzed. These were intercrossed to produce both age-matched homozygous mutant (iv/iv) and control (+/+) mice of the same genetic background. Mean tracheal CBF was 18.9 Hz (SEM = 0.21 Hz, n = 12) in wild-type mice while cilia were static in samples from the mutant cohort (n = 12) with the exception of occasional slow ciliary movement in one individual. The specificity of this defect was confirmed by analyzing the Dnahc11lrm3 (lrm3) mutant, a presumed null allele; lrm3 lacks 50 amino acids from the hinge region and phenocopies the iv situs phenotype [Ermakov et al., 2009]. lrm3 homozgotes (n = 6) showed no ciliary beat in five individuals with one mean value of 18.6 Hz (Fig. 1). Unless stated, the C3H.iv congenic mice were used in all further work.

Figure 1.

CBF values for mouse. Ciliary beat frequency (CBF) determined at 37°C for six independent points on mouse tracheal rings using Fast Fourier transform (FFT) analysis of a digitized photodiode signal from an Olympus inverted microscope. Values for 12 C3H/HeH wild-type (+/+), 12 C3H iv/iv, and six C3H lrm3/lrm3 mice. The observer was blinded to both genotype and gross phenotype.

iv Cilia and Sperm Tails Appear Morphologically Normal

Tracheal ultrastructure of homozygous C3H iv/iv and control C3H mice was visualized using TEM (Fig. 2A and B). Analysis of the data revealed no change in the number of ultrastructural defects in either ciliary microtubules or dynein arms between iv/iv and control samples (n = 4 each, P > 0.5). SEM of trachea from wild-type (C3H) and iv/iv C3H mice revealed extensive fields of cilia in both sample sets (Fig. 2C and D). No systematic differences were apparent in the overall distribution of ciliated cells or the percentage of the airway surface covered by these cells between genotypes or with the age of iv/iv mice (data not shown). Sperm tails from C3H iv/iv mice analyzed by TEM showed no ultrastructural differences from control C3H mice (Fig. 2E–H) and no difference in the number of defects (Chi-squared iv/iv compared to control P = 0.64).

Figure 2.

Cilia and sperm tail ultrastructure–mouse. Cilia ultrastructure of representative mouse airway epithelial cilia (A–D) and sperm tail (E–H) cross-sections examined by TEM (A, B, E–H). Sperm tail cross-sections are at approximately matching points along the tail (E and F, G and H). Representative areas of the mouse airway surface visualized by scanning electron microscopy (SEM) (C, D). C3H/HeH wild-type mouse (A, C, E, G), C3H/HeH iv/iv mouse (B, D, F, H).

iv Sperm Demonstrate Motility Defects Without Affecting Fertility

In light of the similarity between the iv mutant phenotype and aspects of human PCD, we analyzed sperm motility by IVOS. When epidydimal sperm were collected and capacitated, total sperm count was not found to be significantly different between the groups (Fig. 3). However, the total number of motile sperm and the number of sperm showing directed (progressive) motility were statistically lower in iv/iv mice than in controls (Fig. 3; P = 0.028 and P = 0.021, respectively, unpaired t-test). The proportion of offspring weaned from our homozygous iv/iv colony are reduced over controls (data not shown), raising the possibility of sperm-mediated fertility defects. However, a proportion of iv/iv embryos are known to die in utero, during mid or late gestation, due to situs-associated cardiac defects [Icardo, 1990; Icardo and Sanchez de Vega, 1991]. We therefore assessed fertility of iv/iv males by analyzing the number of implanted embryos at 8.5 d.p.c.; this is prior to cardiac-associated lethality. The analysis revealed 10 litters fathered by iv/iv males to give 82 implanted embryos, compared to 87 from wild-type control fathers (Fig. 4). Statistical analysis revealed no significant differences between the sample groups, showing the iv/iv males to be normally fertile.

Figure 3.

Mouse sperm motility. Analysis with IVOS sperm analyser optimized for mouse sperm showing sperm counts (million per ml ± SEM) for C3H wild-type (shaded bars) and C3H iv/iv mice. Each sample was analyzed three times, and five fields counted per reading giving total count of all sperm (total), motile sperm count (motile), and count of sperm with progressive motility (progressive).

Figure 4.

Mouse male fertility. The number of implanted embryos at 8.5 d.p.c. from C3H iv/iv and C3H wild-type sibling control males mated with C57Bl6/J wild-type females. Mean of 10 in each group ± SEM.

iv Mice Demonstrate Chronic Catarrhal Rhinitis and Sinusitis

Consistent with previous reports [Hummel and Chapman, 1959; Layton, 1976], no external differences were observed between iv/iv and control mice. Necropsy analysis demonstrated randomized situs. In the iv/iv C3H mice collected for beat frequency analysis 8/16 were situs solitus, 7/16 situs inversus, and one situs ambiguous. From all iv/iv individuals on all backgrounds, 24/60 were situs inversus or situs ambiguous. Both gross and histological analysis of standard lung sections that include all lobes and major airways revealed no obvious pathology. In a minority of iv/iv and control mice more than 13 weeks old, anatomically normal peribronchiolar lymphoid aggregates were associated with the major airways (Table 1). In particular, there was no evidence of infection or inflammatory disease of the airways (including bronchiectasis), blood vessels, interstitium, or alveoli (data not shown). In contrast, histological analysis of the heads revealed catarrhal rhinitis, characterized by intranasal mucus in the caudal nasal passages variably admixed with neutrophil leukocytes, a phenotype that was fully penetrant in both male and female inbred iv/iv and iv/iv C3H mice (Table 1). Abnormal mucus accumulations in iv/iv mice were distinguishable from anatomically normal nasal mucus that consisted of thin ribbons of mucus on the surface of the brush border in wild type (WT) mice (Fig. 5A-I). Otitis media, another common symptom in PCD patients, was found in 9% of iv/iv mice and was invariably unilateral and characterized by serous effusion and mild thickening of the mucoperiosteum compared with C3H controls (Fig. 5M-P) and the normal contralateral ear (data not shown).

Figure 5.

Catarrhal rhinitis, sinusitis, and otitis media in iv/iv mice. A and B: Caudal nasal passages of 44 week wild-type C3H female control arrow indicates normal mucus; C and D: caudal nasal passages in 5 week iv/iv male arrow indicates abnormal accumulations of bland mucus; E and F: caudal nasal passages in 39 week iv/iv female, arrow indicates increased accumulations of mucus (E), admixed with neutrophil leukocytes, indicated by open arrowhead (F); G and H: mid nasal passages in 44 week wild-type control, arrow indicates normal mucus. I and J: Mid nasal passages in 42 week iv/iv mouse, arrow indicates abnormal mucus and neutrophil leukocytes. K: normal air-filled sinus in a 44 week wild-type female control; L: sinus in a 41 week iv/iv female, arrow indicates serous effusion; M and N: middle ear in 44 week wild-type female, arrowhead indicates normal air-filled bulla, arrow indicates normal mucosa; O and P: otitis media in 41 week iv/iv female, arrowhead indicates serous fluid in bulla, arrow indicates slightly thickened mucosa. Scale bar shown in panel A corresponds to 500 µm in panels A, C, E, G, I, M, O; 100 µm in panels B, D, F, H, J, K, L; 50 µm in panels N, P.

To assess the progressive nature of the PCD-related disease of iv mice, we analyzed the phenotype longitudinally in inbred iv/iv mice. Cohorts of five or more mice were analyzed at 5, 9, 15, and 31–42 weeks of age (Table 1). At 5 weeks, relatively small amounts of bland mucus or mucus containing small numbers of leukocytes were found in the caudal nasal passages of iv/iv mice (Fig. 5C and D). At 31–42 weeks of age, intranasal accumulations of mucus were extensive, admixed with viable and necrotic neutrophil leukocytes (Fig. 5E and F), and were more widely distributed into the mid and rostral snout (Fig. 5I and J). In contrast, sinusitis was not evident in either 5- or 9-week mice while 4/10 and 4/19 mice showed sinusitis at 15 and 31–42 weeks, respectively. (Fig. 5K and L Table 1). The earliest occurrence of otitis media (OM) was at 5 weeks and the incidence of Otitis media did not subsequently increase with age.

DNAH11 Mutations in Patients with PCD

The data from the mouse clearly demonstrate that Dnahc11 mutations can result in immotile tracheal cilia leading to PCD-like phenotypes. Two atypical PCD patients with a clinical history highly suspicious for PCD (Table 2), abnormal ciliary function at 37°C, but normal ciliary ultrastructure were therefore investigated for DNAH11 mutations. Both patients were white Caucasians, unrelated and consanguinity was unlikely. Patient #616 exhibited predominantly static nasal epithelial cilia at 37°C (confirmed on three separate occasions). Occasional slow ciliary activity was measured at one assessment with a mean CBF of 5.26 Hz (SEM = 0.63 Hz). In contrast, patient #730 demonstrated abnormally rapid and dyskinetic ciliary beating (confirmed on two separate occasions). The rapid dyskinetic cilia exhibited erratic interrupted “twitching” and neither performed rhythmical ciliary beating, nor completed a classical range of movement, preventing accurate CBF measurements. TEM analysis of nasal cilia showed no structural differences in either patient compared to normal (Fig. 6A-D), both showing numbers of structural defects within the range of non-PCD samples examined from the clinic (Table 2).

Figure 6.

Cilia ultrastructure–human. Human cilia cross-sections examined by TEM. Normal (A) and cross-section from a “classical” PCD patient with an outer dynein arm (ODA) defect (B), cross-sections from patients #616 (C), and #730 (D) both carrying DNAH11 mutations.

The clinical characteristics and diagnostic investigations are summarized in Table 2. For comparison, we present the clinical characteristics of 27 consecutive patients seen in the Southampton PCD service diagnosed with “classical PCD”. This was defined by abnormal ciliary function plus diagnostic ciliary TEM. Details of 241 consecutive patients in whom PCD was excluded (“non-PCD”) are also included. PCD was excluded on the basis of normal ciliary function (assessed by high-speed video microscopy analysis) and when equivocal, confirmed as normal by further investigation including electron microscopy.

DNA from both patients was screened for mutations of DNAH11. Two sequence variations were detected in patient #730 (Fig. 7). A nonsense mutation in exon 53, c.8719C>T (g.21755845C>T NCBI36/hg18), changes arginine 2907 (in the AAA4 domain after the 4th P-loop) into a stop codon (p.Arg2907*). A missense mutation in exon 47, c.7793C>T (g.21744970C>T NCBI36/hg18), changes the highly conserved proline 2598 in the third AAA domain, to leucine (p.Pro2598Leu). This variation is not present in dbSNP or control samples and is identified as “probably damaging” by Polyphen (its highest score). In patient #616, only one mutation was detected (Fig. 7), a nonsense mutation in exon 39 (c.6527C>A, g.21711640C>A NCBI36/hg18) changing serine 2176 into a stop codon (p.Ser2176*), positioned in AAA2, just before the second P-loop. Sanger sequencing of amplified exons does not detect heterozygous deletions, hence deletion of one or more exons cannot be ruled out.

Figure 7.

Novel mutations found in this study. A–C: Chromatograms of novel mutations found in this study. D: Schematic of DNAH11/Dnahc11 protein showing location of mutations from this study (blue) and previous publications from mouse (green) and human (black) relative to the recognized dynein heavy chain domains. Mutations causing truncation or frame shift (FS) are shown with a dotted line downstream of the mutation. From the N-terminus mutations are from the following studies c.855–1G>A [Pifferi et al., 2010]; p.Trp1382* [Pifferi et al., 2010]; lrm3 mouse–50 amino acids missing [Ermakov et al., 2009]; p.Ser2176* (this study); iv mouse [Supp et al., 1997]; p.Pro2598Leu (this study); p.His2712Arg [Pifferi et al., 2010];p.Arg2852* [Bartoloni et al., 2002]; p.Arg2907* (this study); p.Lys3038Thrfs*13 [Berg et al., 2011]; p.Gly3428Arg [Pifferi et al., 2010]; p.Tyr4128* [Schwabe et al., 2008]; p.4518 4523delinsQ [Schwabe et al., 2008].


This study has identified the iv mouse as a viable model for aspects of the human disease PCD. This mouse line has been constantly bred as a homozygous colony for over 50 years, demonstrating that it is robust, viable, and that it demands only normal husbandry, in contrast to the viability issues seen in other models including Dnahc5 [Tan et al., 2007], nm1054 [Lee et al., 2008] and the complex preparatory considerations associated with the Dnaic conditional mutant [Ostrowski et al., 2010]. We have not only confirmed that the iv model has static respiratory cilia at 37°C, but that it develops PCD-related disease including rhinitis, sinusitis, and otitis media. Consistent with other PCD mouse models [Ibanez-Tallon et al., 2002; Lee et al., 2008; Tanaka et al., 2004], none of the mice in this study developed significant lung pathology. This is perhaps an age-related phenomenon (our oldest mice were 42 weeks old), or lack of disease may be associated with the relatively sterile environment of the animal facilities as suggested for CF models [Davidson and Rolfe, 2001]. The mice were maintained specific pathogen free, so further work will be required to establish whether introduction of respiratory pathogens results in lower airway disease. Sinusitis and otitis media were seen in mouse mutants lacking the Wnt inhibitor Cby1 (MIM# 607757) that were unable to clear bacteria from the sinuses upon intranasal bacterial challenge leading to upper airway inflammation, however additional morphological pulmonary defects were also evident from shortly after birth [Love et al., 2010]. Strikingly, lower airway disease, including pneumonia, was reported for Spag6 (MIM# 605730), Spag16 (MIM# 6121736) double mutants, however mortality is very high with 100% lethality by 5 weeks of age, making it far more severe than human PCD [Zhang et al., 2007]. It is tempting to speculate that it is the presence of chronic infection in man, taking place over a period of years rather than months, which is responsible for bronchiectasis and other lung pathology rather than a direct link to dyskinesia.

There was evidence of decreased progressive sperm motility in the iv mice, but since the line has been successfully bred from homozygotes for many years, any associated subfertility cannot be considered a major problem with this mutation, a conclusion supported by the results of the fertility test. Although male infertility is said to be common in PCD, it is not universal [Munro et al., 1994]. Indeed, one of the DNAH11 PCD patients reported by Schwabe [Schwabe et al., 2008] had apparently fathered two children without medical assistance. This might suggest that this dynein is normally absent in sperm, although the conservative changes seen in mouse sperm motility argue that there is more likely to be additional functional redundancy in the sperm.

This study contributes to the evolving story concerning DNAH11 as a cause of PCD. Mutations in DNAH11 have to date been described in a family with PCD [Schwabe et al., 2008] associated with a sibling pair and in two isolated cases [Bartoloni et al., 2002; Pifferi et al., 2010]. The static cilia in our mouse model, contrasting with previously described human PCD cases, led us to investigate a patient (#616) with consistently static cilia, normal ciliary ultrastructure, and a typical clinical PCD phenotype. A second patient (#730) with PCD phenotype, normal ciliary ultrastructure and stiff, hyperfrequent cilia had two mutations in DNAH11, consistent with this being the primary defect. We were only able to identify one mutation in patient #616, although both parents are asymptomatic and it is therefore unlikely to be a simple dominant mutation. The entire coding sequence, the 5′ and 3′ UTRs, and the intronic regions flanking each exon were ruled out. The promoter, which is not well described yet, and regulatory regions were not screened. A second DNAH11 mutation resulting from deletion of one or more exons would not be evident from normal DNA sequencing and remains a possibility. Similarly allele dropout due to a mutation in the primer binding site cannot be completely excluded. The prospect that the patient carries mutations in another PCD gene must also be considered with the DNAH11 mutation contributing to a multigenic defect in combination with one or more other PCD locus mutations. A recent report of a PCD patient with only a single frameshift mutation in DNAH11 was suggested to result from digenic inheritance with a DNAH2 (MIM# 603333) variant [Berg et al., 2011], although the authors indicate that further studies are required. However, these human data, combined with the evidence that mutations in the mouse homolog can be associated with static cilia, supports the hypothesis that DNAH11 mutations can cause static cilia, in addition to the previously described association with stiff vibrating/hyperfrequent cilia. Mutations in this gene should therefore be considered in patients with a PCD phenotype associated with either static or hyperfrequent cilia.

The three DNAH11 mutations that we have identified seem highly likely to affect protein function. Two are truncations resulting from premature stop codons and one is the substitution of a highly conserved nonhydrophobic proline with a hydrophobic leucine within the third AAA domain (p.2598Pro>Leu). Prior to this study, all mutations identified in DNAH11 (Fig. 7) have been associated with hyperfrequent ciliary beating. Mutations in genes resulting in outer dynein arm (ODA) defects (as determined by TEM) generally have a static ciliary phenotype [Chilvers et al., 2003]. As ODAs are present in DNAH11 and Dnahc11 mutants (as assessed by TEM), it seems possible that this dynein may have an auxiliary or regulatory role, resulting in altered mechanical characteristics of the axoneme if the molecule is absent or substantially truncated. However, the basis of the immotile cilia in patient #616 remains difficult to ascertain as only a single truncated allele has so far been identified. In contrast, the iv and lrm3 mouse lines show immotile tracheal cilia from presumed null mutations [Ermakov et al., 2009; Supp et al., 1999] arguing that the true null phenotype is immotile cilia. Further study will be required to determine whether mouse and human cilia truly have identical requirements for DNAH11/Dnahc11 function.

A model of a human genetic disease can seek to recreate the mutation, the physiological defect, defects at the cellular level, or all of these. On occasions all outcomes will occur in combination, but this is not always the case. The iv mouse model clearly recreates the physiological phenotype of immotile respiratory cilia through a Dnahc11 mutation. It therefore should be useful for modeling and therapy development associated with (1) loss of DNAH11 function, (2) loss of respiratory cilia motility, and (3) loss of cilia-driven clearance from the lung. We have identified a viable and readily accessible mouse model of PCD that will allow future studies into therapeutic approaches including, but not limited to, gene therapy. It will allow the hypothesis that recurrent infection, rather than PCD itself, underlies bronchiectasis to be tested. Importantly, it will allow PCD and putative therapies to be investigated from birth into old age, something not possible with current models. Moreover, we have highlighted important implications for patient diagnosis. TEM is often quoted as the gold standard test in the diagnostic work-up of PCD. This study contributes to the growing evidence that PCD will be missed in a proportion of patients with normal ciliary architecture if ciliary function studies are not routinely performed. Indeed some studies have suggested that normal electron microscopy is as common as 10–15% in PCD. To date DNAH11 is the only gene with mutations associated with PCD that has been described in patients with normal TEM and the iv mouse models this.


Our thanks to the patients and their families for their participation in this study. The staff of Biomedical Imaging Unit Southampton provided technical support and assistance. Adele Austin, Caroline Barker, Jenny Corrigan, Liz Darley at MRC Harwell for histology support. Staff in the Mary Lyon Centre for animal care. The Gordon Research Conference on Cilia, Mucus, and Mucociliary Interactions for introducing the authors to one another.