Human Molecular Genetics, 2002, Vol. 11, No. 6 715-721
© 2002 Oxford University Press
Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus
Howard Hughes Medical Institute, Laboratory of Molecular Biology, The Rockefeller University, New York, NY 10021, USA
Received January 21, 2002; Accepted January 24, 2002.
DDBJ/EMBL/GenBank accession no. AF466704.
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ABSTRACT |
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Primary ciliary dyskinesia (PCD), also known as Kartagener’s syndrome, is a human syndrome that results from ciliary dysfunction. This syndrome is characterized by recurrent respiratory infections, situs inversus and infertility. In some cases, hydrocephalus is also observed. We have characterized an insertional mutation in a mouse axonemal dynein heavy chain gene (Mdnah5) that reproduces most of the classical features of PCD, including recurrent respiratory infections, situs inversus and ciliary immotility. These mice also suffer from hydrocephalus and die perinatally. Electron microscopic studies demonstrate the loss of axonemal outer arms. These results show that mutations in Mdnah5 are a primary cause of PCD and provide direct evidence that mutations in an axonemal dynein can cause hydrocephalus. Mutations in the human DNAH5 have recently been identified in PCD patients. Comparison of the mouse model and the human data suggests that the degree of ciliary dysfunction is causally related to the severity of human PCD, particularly the presence of hydrocephalus.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Primary ciliary dyskinesia (PCD), also known as Kartagener’s syndrome, is a human disorder (OMIM 244400) that results from ciliary dysfunction and is characterized by randomization of left–right asymmetry, recurrent respiratory infections and infertility (1–3). In several reported cases hydrocephalus was also associated with PCD (4–8). PCD is a genetically and clinically heterogeneous disorder. Linkage studies have revealed extensive locus heterogeneity in PCD (9,10), consistent with the fact that patients display a number of axoneme deficiencies, including defective nexin links and radial spokes, disordered or absent microtubules and inner and outer dynein arm abnormalities (11).
The outer and inner dynein arms of the axoneme provide the motor force during ciliary bending. Dynein arms are formed by assembly of several light, intermediate and heavy chain axonemal dyneins. The axonemal structure is highly conserved from lower to higher eukaryotes and is extensively studied in the flagellate alga Chlamydomonas. There at least 16 dynein heavy chain isozymes in eukaryotes (12). Recently, there has been a renewed interest in vertebrate axonemal function since axonemal dynein deficiencies have been implicated in randomization of left–right patterning and in PCD (13–15). Two described mice lines deficient in axonemal dynein chains have been proposed as mouse models for PCD. Mice lacking Mdhc7 had reduced sperm motility (16) and lrd mutant mice had random left–right patterning (17). None of these models presented all of the symptoms associated with the human disorder.
In this study we generated axonemal dynein heavy chain 5 (Mdnah5) deficient mice that lack outer dynein arms and exhibit cilia immotility, random left–right axis specification, chronic respiratory infections and hydrocephalus. This mouse model has most of the pathological features associated with PCD. More importantly, this mouse model is relevant for further studies of the human syndrome, since mutations in eight families affected with PCD have just been reported in the human homolog of Mdnah5 (15). The mouse model also provides first direct evidence that loss of function of an axonemal dynein can cause hydrocephalus, and suggests that, in some cases, human PCD with associated hydrocephalus may result from null mutations in the DNAH5 gene.
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RESULTS |
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Generation of Mdnah5–/– deficient mice
Mice with an insertional mutation in the Mdnah5 gene were generated while establishing a transgenic line for an unrelated gene. We observed that mice homozygous for the transgene developed a phenotype that was not expected from the transgene expression and was not detected in any of four other transgenic lines, suggesting that the transgene insertion had disrupted another gene. We identified the sequence flanking the transgene integration site and found that it encompasses a novel gene encoding an axonemal dynein heavy chain protein (Mdnah5), which is a structural component of cilia. The Mdnah5 gene is the homolog of human DNAH5 (15,18). We determined Mdnah5 full-length cDNA from genomic sequence using homology to other axonemal dyneins such as Dnahc8 (19) and the
heavy chain of Chlamydomonas flagellar outer arm dynein (20). Mdnah5 has 79 exons and is located on murine chromosome 15 spanning over 
250 kb (Fig. 1A). It contains an open reading frame encoding 4621 amino acid residues. The encoded protein contains all conserved domains of heavy chain dyneins, including six P-loops for ATP hydrolysis and a microtubule-binding site (Fig. 1D). Analysis of the 5' and 3' regions flanking the transgene insertion revealed that the transgene integrated between exon 16 and exon 22 of Mdnah5, deleting exons 17–21 (Fig. 1B and C). RT–PCR amplification detected a transcript of Mdnah5 with the transgene inserted between exons 16 and 22, resulting in the generation of a stop codon at the start of the transgene. The predicted translated product of this fusion transcript lacks all of the known functional domains of the protein (Fig. 1D). These data demonstrate that the transgene insertion caused a simple chromosomal deletion that resulted in the disruption of the Mdnah5 gene and truncation of Mdnah5 protein, producing a functional null mutation.
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Mdnah5–/– mice present randomization of left–right asymmetry, recurrent respiratory infections and inner ear infections
PCD, or Kartagener’s syndrome, is a human syndrome caused by ciliary dysfunction and characterized by situs inversus, chronic infection of the respiratory tract and infertility (1,2). Detailed pathological examination of Mdnah5–/– mice revealed most of the features that characterize human PCD. Inspection of the internal viscera of Mdnah5–/– mice demonstrated random left–right axis specification (Fig. 2A and B). Homozygous animals displayed either complete (both dextrocardia and reversal of the abdominal organs) (Fig. 2A), partial (Fig. 2B) or no situs inversus. Obvious signs of respiratory distress and a failure to respond to noise were also noted (data not shown). Analysis of paranasal sinuses showed that excess mucus, containing numerous white blood cells, accumulated in the nasal sinuses of mutant animals (Fig. 2D and E). Examination of the middle ear also revealed severe infection in the timpanic cavity of Mdnah5–/– mice (Fig. 2G and H). These findings are consistent with the recurrent respiratory infections and otitis that are associated with human PCD.
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Mdnah5–/– mice develop hydrocephalus
In addition to these common features associated with PCD, all Mdnah5–/– mice suffered from hydrocephalus (Fig. 3). Hydrocephalus was detected as early as 3–5 days after birth and developed to severe enlargement of the lateral ventricles leading to thinning of the cerebral cortex, multiple hemorrhages and compression of the cerebellum (Fig. 3C and D). Mdnah5–/– mice also showed growth retardation (Fig. 3A and B), early mortality and abnormal gait (Supplementary Material). It was not possible to determine whether Mdnah5–/– mice had infertility, a common feature of Kartagener’s syndrome, since most of them die during the first 2–3 weeks of age. No abnormalities were noted in heterozygous littermates, suggesting that this mutation is fully recessive.
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The cilia of Mdnah5–/– mice are immotile and lack outer arms
As in Kartagener’s syndrome, ciliary dysfunction might cause the recurrent infections, situs inversus and hydrocephalus observed in these animals. Therefore we further analyzed the motility and structure of the cilia in Mdnah5–/– mice. Tissue slice cultures were prepared from brain ventricles and trachea of mutant and wild-type mice and assayed for ciliary motility by videomicroscopy. Cilia of wild-type mice displayed the characteristic beating motion (Supplementary Material), whereas those from mutant mice were completely immotile (Supplementary Material). Ciliary beating originates from the sliding of axonemal microtubules due to the ATP-dependent dynein-generated force. The outer and inner arms that interlink the microtubule doublets are formed by the concerted assembly of heavy, intermediate and light chain dyneins (12,21) To assess whether Mdnah5 is required for dynein arm assembly we analyzed nasal epithelium samples from Mdnah5–/– mice by electron microscopy. Dynein outer arms were completely absent in the cilia of mutant mice (Fig. 4), providing a structural explanation for the immotility of Mdnah5–/– cilia. Thus, as in human PCD, the pathological features observed in Mdnah5–/– mice can be explained by cilia immotility. Lack of ciliary clearance of the respiratory tracts and Eustachian tubes causes the recurrent infections (22), and deficient motility of the nodal cilia causes the randomization of the left–right axis specification in these mutant mice (23,24). Similarly, the failure of ependymal cilia to support the flow of cerebral spinal fluid in Mdnah5–/– mice results in increased ventricular volumes, brain damage and perinatal death.
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DISCUSSION |
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Recently, it was proposed that the leftward flow generated by the monocilia of the embryonal node creates an asymmetrical distribution of one or more morphogens that trigger the genetic cascade that differentiates the left and right sides (23,24). This is supported by studies in mice that lack nodal cilia, such as Kif3A–/–, Kif3B–/– and Tg737 mutants (25–27) or mice that have immotile nodal cilia, such as lrd–/– and Hfh4–/– animals (17,28). The randomization of left–right specification in Mdnah5–/– mice due to the truncation of Mdnah5, and expression of Mdnah5 in the embryonic node (15) also support the nodal-flow hypothesis. Further analysis of the relationship between Mdnah5, lrd, Kif3A, Kif3B, Tg737, Hfh4 and other factors involved in ciliogenesis will be required to elucidate their role in normal left–right patterning of the mammalian embryo.
PCD is a genetically and clinically heterogeneous disorder. So far mutations have been found in only two genes, dynein intermediate chain DNAI1, in four families (13,14) and DNAH5, the human homolog of Mdnah5, in eight families (15). Disruption of the Mdnah5 gene results in a mouse phenotype that replicates most of the features of classic Kartagener’s syndrome, and also displays hydrocephalus. In the study of Olbrich et al. (15) no hydrocephalus was reported (although brain ventricular volumes were not measured). There are several reported cases of PCD patients that have evident hydrocephalus (4–8). In one case the histologic analysis of the nasal cilia from the patient revealed a total loss of outer dynein arms (4), as in Mdnah5 mutant mice. A possible interpretation of these findings is that mutations of the DNAH5 gene in humans can cause PCD with associated hydrocephalus, but that the presentation of the disease is dependent on the severity of the mutation and the specific genetic background of the patient. Nine out of the 10 mutations in DNAH5 identified in human PCD affected patients are located within the C-terminal two-thirds of the protein (15). One truncation maps close to the N-terminus, similar to the truncation we have identified in Mdnah5 mice. However, patients carrying this truncation are compound heterozygotes, and carry a second allele with a less severe mutation. Studies in Chlamydomonas have shown that a dimerization domain is probably located within the N-terminal third of dynein heavy chains and that axonemal dynein mutants having a truncation after this domain can still partially assemble outer arm structures (29). The truncation at exon 17 in Mdnah5 mice most likely results in the absence of this domain in homozygous mice. Consistent with the mouse data, we suggest that lack of this dimerization domain in DNAH5 alleles in humans would result in PCD with associated hydrocephalus (4), and that alleles with downstream truncations might result in a syndrome closely resembling PCD (15). In a general sense, this is consistent with the genetic heterogeneity of PCD in humans, and with previous mouse models of PCD that display only situs inversus (17) or infertility (16,19), since the specific clinical features or phenotypes resulting from mutations in ciliary proteins will depend on the degree of residual function retained in the mutant protein, and its sites and levels of expression in vivo. This hypothesis could be investigated further with transgenic rescue experiments introducing different truncation mutants in Mdnah5 mice, providing further insight into the etiology of PCD and hydrocephalus in humans. In addition, these experiments will facilitate the identification of functional domains of axonemal dynein heavy chains.
Indirect evidence that ciliary dysfunction can lead to hydrocephalus has been obtained previously. Partial penetrance of hydrocephalus was described in mice deficient in the transcription factor Hfh4, which was proposed to regulate lrd and other dyneins (28). In Tg737 mice, the observed partial loss of cilia in the brain ventricles could have resulted as a primary consequence of the mutation, or a secondary effect of increased intracranial pressure (27). While ciliary damage is acknowledged as a common secondary effect of hydrocephalus in humans due to increased intracranial pressure, the Mdnah5–/– mouse model provides direct evidence that ciliary dysfunction can be a primary cause of hydrocephalus. This is consistent with clinical reports describing hydrocephalus in patients with ciliary dysfunction and unknown genetic etiology (4–8).
Several mouse lines with axonemal dynein deficiencies have been proposed previously as models for PCD. However, these models present a very limited subset of the classical symptoms associated with the human disorder. For example, mice deficient in axonemal dynein Mdhc7 displayed reduced sperm motility (16), and lrd mice demonstrated randomization of the left–right body axis (17). In contrast, the mouse model described here presents most of the clinical features of the human syndrome, such as immotile cilia, randomization of left–right asymmetry, chronic respiratory and inner ear infections and hydrocephalus. Our data establish that the Mdnah5 gene is a critical component of the outer dynein arm of mammalian cilia, that its disruption can lead to PCD and hydrocephalus, and that screens for mutations in this gene in human patients with PCD or hydrocephalus may lead to genotype/phenotype correlations that provide insight into both the function of this dynein and the severity of the clinical features resulting from those mutations.
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MATERIALS AND METHODS |
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Characterization of the transgene integration site and genomic organization of the Mdnah5 gene
The genomic sequence upstream of the transgene integration site was determined using the VectoretteTM System (Sigma/Genosys). Genomic DNA from transgenic mice was digested with several restriction enzymes and ligated into corresponding Vectorette Units according to the manufacturer’s instructions. PCR amplification using transgene- and vectorette-specific primers yielded a 1.4 kb band and a 0.5 kb band from AciI and RsaI Vectorette constructs, respectively, which partially overlapped. Genomic sequence obtained from sequencing these bands was BLASTed against the Celera Mouse Genome database. The 20 kb genomic region corresponding to the 5' transgene integration site was masked using RepeatMasker (A.F.A.Smit and P.Green; http://repeatmasker.genome.washington.edu/RM/RepeatMasker.html) and BLASTed against the human genome, revealing stretches of 85% similarity with regions on chromosome 5p15 where DNAH5 has been mapped (18).
The exon–intron structure and amino acid sequence of Mdnah5 were deduced from mouse and human genomic sequences using GenomeScan (30) based on similarity to mouse axonemal dynein Dnahc8 (19) and to the heavy chain of Chlamydomonas flagellar outer arm dynein (20), which has the highest sequence similarity to Mdnah5 (60% similarity and 42% identity) out of the known 16 dynein heavy chains in Chlamydomonas (12). Certain regions of Mdnah5 were confirmed by RT–PCR and sequencing using the following primers: 2F, 5'-TGTTTGCAATTGTAGCTTCCTG-3' and 5R, 5'-CATCTGCCGTGTCTAACGTG-3'; 16F, 5'-CTTGATTGAGTTCCGCATCC-3' and 19R, 5'-TCCACTGTCTAACCCCCTTG-3'. Exons 14, 19, 22 and 75 were PCR amplified from genomic DNA using the following primers: 14F, 5'-CCAGAATTTTGCGACTGACA-3' and 14intR, 5'-TGCTGTGTGACTTTCTCACG-3' to amplify exon 14; 19F, 5'-GTGTTAGAGGAAGCCCGTGA-3' and 19intR, 5'-GCCGTCAATCGCTTATCATT-3' to amplify exon 19; 22F, 5'-ACTTACCCATCCCAGTGCAA-3' and 22intR, 5'-TGTCACCTCGGTCTGAATTG-3' to amplify exon 22; 75F, 5'-ATGACAGCCCAGAGGTGTTT-3' and 75intR, 5'-AACTGGTCCTTCACTCTGACAA-3' to amplify exon 75. Exons 14, 19, 22 and 75 were RT–PCR amplified from RNA using the Superscript RT–PCR system (Gibco BRL) and the following primers: 14F and 15R, 5'-TGCAAACGGTGACACAGGTA-3'; 19F and 20R, 5'-TCCACTGTCTAACCCCCTTG-3'; 22F and 23R, 5'-AAGTCGGTGGAGATCTGCTG-3'; 75F and 76R, 5'-TCTGGCATCAAACATGCAAT-3'. The 5' and 3' fusion sites flanking the transgene in the cDNA of Mdnah5 were RT–PCR amplified and sequenced using primer 14F and a 5' transgene-specific primer to amplify the 5' fusion site, and 22R, 5'-TTGCACTGGGATGGGTAAGT-3' and a 3' transgene-specific primer to amplify the 3' fusion site. The genomic sequence downstream of the transgene, integration site was determined by PCR amplification and sequencing using a transgene-specific primer at the 3' end of the transgene, and the primer 21intR (5'-TCCTCCCAGAGGAGTCAGAG-3') in intron 21 of Mdnah5.
Generation of Mdnah5–/– mice
The transgene was injected into C57BL/6xCBA/J fertilized oocytes using standard procedures. Mice were maintained in the same C57BL/6 inbred hybrid genetic background. Intercrossing of heterozygous transgenic mice (13 pairs) yielded 69% (248 of 361) positive transgenic offspring. Hydrocephalic mice tested positive for the transgene and accounted for 20% of the progeny (73 of 361). Mouse genotyping was performed initially by Southern blotting and later by PCR with transgene-specific primers (Fig. 1C).
Morphology and histology
Mice were anesthetized with Nembutal Sodium Solution (Abbott) and perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde/PBS. Dissected tissue samples were kept in 4% paraformaldehyde/PBS overnight and in some cases were decalcified in 8% EDTA, 0.1 M cacodylate and 5% sucrose for 2 days. Paraffin embedding was performed by sequential incubations in 70% ethanol, 95% ethanol, butanol and paraffin wax (Tissueprep2, Fisher Scientific). Paraffin-embedded tissues were sectioned at 10 µm on a paraffin microtome (HM 335E, Microm). Sections were hydrated and stained with 1/20 dilution of Giemsa stain 0.4% solution (Sigma), dehydrated and mounted on microscope slides with Permount (Fisher Scientific). Images were acquired with an Axioscop2 (Zeiss) microscope.
Electron microscopy
Ciliary epithelium of nasal mucosa was dissected from mice sequentially perfused with saline and 4% paraformaldehyde/0.5% glutaraldehyde in PBS. Nasal epithelia samples were then incubated in 2.5% glutaraldehyde at 4°C overnight. Tissue specimens were fixed by conventional methods (OsO4), treated in 1.5% tannic acid and processed for electron microscopy following standard procedures (31).
Cilia motility analysis
Brain sagittal sections and trachea cross-sections of 100 µm thickness were prepared and incubated in L-15 medium (Specialty Media) at room temperature in culture slides (Supercell, Fisher Scientific). Cilia motility was monitored with a digital photographic camera connected to an Axioscop2 (Zeiss) microscope.
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SUPPLEMENTARY MATERIAL |
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Supplementary Material is available at HMG Online.
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ACKNOWLEDGEMENTS |
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We would like to thank the Rockefeller Transgenic Service Laboratory, Helen Shio of the Bioimaging Facility, Niels Adams, Christopher Wynder and Achim Jungbluth for technical assistance, and Daniel Besser for scientific advice and critical reading of the manuscript. This research was supported by the Howard Hughes Medical Institute, NIH NINDS P01 NS30532; AT Children’s Project and HHMI (I.I.-T.) and by NIH MSTP grant GM07739 (S.G.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 212 327 7956; Fax: +1 212 327 7878; Email: heintz@rockefeller.edu
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REFERENCES |
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K.-F. Lechtreck, P. Delmotte, M. L. Robinson, M. J. Sanderson, and G. B. Witman Mutations in Hydin impair ciliary motility in mice J. Cell Biol., February 6, 2008; 180(3): 633 - 643. [Abstract] [Full Text] [PDF]
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K.-F. Lechtreck and G. B. Witman Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility J. Cell Biol., February 12, 2007; 176(4): 473 - 482. [Abstract] [Full Text] [PDF]
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S. H. Randell, R. C. Boucher, and for the University of North Carolina Virtual Lung Effective Mucus Clearance Is Essential for Respiratory Health Am. J. Respir. Cell Mol. Biol., July 1, 2006; 35(1): 20 - 28. [Full Text] [PDF]
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M. Merchant, M. Evangelista, S.-M. Luoh, G. D. Frantz, S. Chalasani, R. A. D. Carano, M. van Hoy, J. Ramirez, A. K. Ogasawara, L. M. McFarland, et al. Loss of the Serine/Threonine Kinase Fused Results in Postnatal Growth Defects and Lethality Due to Progressive Hydrocephalus Mol. Cell. Biol., August 15, 2005; 25(16): 7054 - 7068. [Abstract] [Full Text] [PDF]
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H. Tanaka, N. Iguchi, Y. Toyama, K. Kitamura, T. Takahashi, K. Kaseda, M. Maekawa, and Y. Nishimune Mice Deficient in the Axonemal Protein Tektin-t Exhibit Male Infertility and Immotile-Cilium Syndrome Due to Impaired Inner Arm Dynein Function Mol. Cell. Biol., September 15, 2004; 24(18): 7958 - 7964. [Abstract] [Full Text] [PDF]
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A. L. M. Ferri, M. Cavallaro, D. Braida, A. Di Cristofano, A. Canta, A. Vezzani, S. Ottolenghi, P. P. Pandolfi, M. Sala, S. DeBiasi, et al. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain Development, August 1, 2004; 131(15): 3805 - 3819. [Abstract] [Full Text] [PDF]
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E. Bonnafe, M. Touka, A. AitLounis, D. Baas, E. Barras, C. Ucla, A. Moreau, F. Flamant, R. Dubruille, P. Couble, et al. The Transcription Factor RFX3 Directs Nodal Cilium Development and Left-Right Asymmetry Specification Mol. Cell. Biol., May 15, 2004; 24(10): 4417 - 4427. [Abstract] [Full Text] [PDF]
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S. L. Brody Genetic Regulation of Cilia Assembly and the Relationship to Human Disease Am. J. Respir. Cell Mol. Biol., April 1, 2004; 30(4): 435 - 437. [Full Text] [PDF]
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M. Zariwala, W. K. O'Neal, P. G. Noone, M. W. Leigh, M. R. Knowles, and L. E. Ostrowski Investigation of the Possible Role of a Novel Gene, DPCD, in Primary Ciliary Dyskinesia Am. J. Respir. Cell Mol. Biol., April 1, 2004; 30(4): 428 - 434. [Abstract] [Full Text] [PDF]
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B. R. Grubb, J. H. Jones, and R. C. Boucher Mucociliary transport determined by in vivo microdialysis in the airways of normal and CF mice Am J Physiol Lung Cell Mol Physiol, March 1, 2004; 286(3): L588 - L595. [Abstract] [Full Text] [PDF]
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B. E. Davy and M. L. Robinson Congenital hydrocephalus in hy3 mice is caused by a frameshift mutation in Hydin, a large novel gene Hum. Mol. Genet., May 15, 2003; 12(10): 1163 - 1170. [Abstract] [Full Text] [PDF]
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I. Ibanez-Tallon, N. Heintz, and H. Omran To beat or not to beat: roles of cilia in development and disease Hum. Mol. Genet., April 2, 2003; 12(90001): R27 - 35. [Abstract] [Full Text] [PDF]
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G. K. H. Przemeck, U. Heinzmann, J. Beckers, and M. Hrabe de Angelis Node and midline defects are associated with left-right development in Delta1 mutant embryos Development, January 1, 2003; 130(1): 3 - 13. [Abstract] [Full Text] [PDF]
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