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Original research
Truncating mutations in exons 20 and 21 of OFD1 can cause primary ciliary dyskinesia without associated syndromic symptoms
  1. Zuzanna Bukowy-Bieryllo1,
  2. Alicja Rabiasz1,
  3. Maciej Dabrowski1,
  4. Andrzej Pogorzelski2,
  5. Alina Wojda1,
  6. Hanna Dmenska3,
  7. Katarzyna Grzela4,
  8. Jakub Sroczynski5,
  9. Michal Witt1,
  10. Ewa Zietkiewicz1
  1. 1 Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
  2. 2 Rabka Branch, Institute of Tuberculosis and Lung Diseases, Rabka-Zdroj, Poland
  3. 3 Department of Lung Physiology, Children’s Memorial Health Institute, Warsaw, Poland
  4. 4 Departments of Pulmonology and Allergy, Warsaw Medical University, Warsaw, Poland
  5. 5 Department of Paediatric Otolaryngology, Poznan University of Medical Sciences, Poznan, Wielkopolskie, Poland
  1. Correspondence to Dr Zuzanna Bukowy-Bieryllo, Institute of Human Genetics, Polish Academy of Sciences, Poznan 60-479, Poland; zuzanna.bukowy-bieryllo{at}


Background Primary ciliary dyskinesia (PCD) is a motile ciliopathy, whose symptoms include airway infections, male infertility and situs inversus. Apart from the typical forms of PCD, rare syndromic PCD forms exist. Mutations of the X-linked OFD1 gene cause several syndromic ciliopathies, including oral-facial-digital syndrome type 1, Joubert syndrome type 10 (JBTS10), and Simpson-Golabi-Behmel syndrome type 2, the latter causing the X-linked syndromic form of PCD. Neurological and skeletal symptoms are characteristic for these syndromes, with their severity depending on the location of the mutation within the gene.

Objectives To elucidate the role of motile cilia defects in the respiratory phenotype of PCD patients with C-terminal OFD1 mutations.

Methods Whole-exome sequencing in a group of 120 Polish PCD patients, mutation screening of the OFD1 coding sequence, analysis of motile cilia, and magnetic resonance brain imaging.

Results Four novel hemizygous OFD1 mutations, in exons 20 and 21, were found in men with a typical PCD presentation but without severe neurological, skeletal or renal symptoms characteristic for other OFD1-related syndromes. Magnetic resonance brain imaging in two patients did not show a molar tooth sign typical for JBTS10. Cilia in the respiratory epithelium were sparse, unusually long and displayed a defective motility pattern.

Conclusion Consistent with the literature, truncations of the C-terminal part of OFD1 (exons 16–22) almost invariably cause a respiratory phenotype (due to motile cilia defects) while their impact on the primary cilia function is limited. We suggest that exons 20–21 should be included in the panel for regular mutation screening in PCD.

  • oral-facial digital syndrome type 1
  • primary ciliary dyskinesia
  • syndromic PCD
  • Joubert syndrome type 10
  • Simpson-Golabi-Behmel syndrome type 2
  • ciliopathy
  • motile cilia biogenesis

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Ciliopathies are diseases caused by inherited defects in the genes involved in the biology of cilia.1 The majority of ciliopathies are syndromic disorders caused by the dysfunction of primary cilia, organelles present on the surface of most of the cells in the human body. Primary ciliary dyskinesia (PCD) (MIM244400), with the prevalence estimated at approximately 1 in 20 000 live births,1 2 is the only ciliopathy caused by the dysfunction of motile cilia. The symptoms observed in PCD patients are usually limited to tissues in which motile cilia are present: dysmotility of cilia in the respiratory epithelium causes impaired mucociliary clearance and recurrent respiratory infections leading to recurrent sinusitis, rhinitis and bronchiectasis; dysfunction of flagella in sperm cells leads to male infertility; impaired motility of cilia present in the embryonic node causes laterality defects in approximately half of the patients.1

To date, approximately 40 genes causing the disease have been found.1 In most of PCD cases, the disease is inherited as an autosomal recessive trait. The X-linked inheritance of PCD has also been reported in male patients with pathogenic variants in three genes: RPGR, OFD1 or PIH1D3.3–6 Mutations of RPGR and OFD1, in addition to causing classical PCD clinical symptoms due to motile cilia impairment, also affect primary cilia and lead to syndromic forms of PCD.3 4 7 8

The OFD1 gene, localised in the short arm of chromosome X (p22.2), encodes a 1012 amino acid protein, a component of centrioles and basal bodies, essential for the biogenesis of both primary and motile cilia.9–13 OFD1 protein contains several functional domains (figure 1A): the N-terminally located microtubule-binding LisH domain (amino acid positions 72–101, exon 3); and seven protein–protein binding coiled-coil domains (CC1–CC7) spread from the middle of the protein to its C-terminus.13 14 The C-terminal part of the protein (amino acids 719–991, exons 16–23) is predicted to contain five intrinsically disordered regions (IDRs), which are involved in protein–protein interactions15–17 (figure 1A). Under native conditions, IDRs have no single predicted tertiary structure.18

Figure 1

Genomic changes in OFD1 identified in the patients. (A) Exonic structure of OFD1 protein and its domains. The range of location of mutations causing three syndromes, OFDS1, SGBS2 and JBTS10, is indicated above the exons. Untranslated regions (UTRs) in exons 1 and 23 are indicated by light grey; the N-terminally located microtubule-binding LisH domain is marked with orange diamond. (B) Pathogenic OFD1 variants identified in the Polish PCD patients. Upper panel: pedigrees of the Polish PCD patients with pathogenic variants in OFD1. Numbers below the symbols indicate genotyped individuals and their genotypes. Filled symbols—hemizygous males; black dot inside the circles indicates a carrier female, grey dot (family 440) indicates carrier status based on data from external diagnostic laboratory. Absence of the dot in family 259 indicates lack of the pathogenic allele in the mother. Lower panel: pathogenic variants and chromatograms of the respective exon sequence in wild-type individuals, hemizygous males (hemiz) and heterozygous carriers (hetero). (C) RT-PCR analysis of OFD1 transcripts expression level for patient 581 and two non-PCD controls. Upper part: PCR bands of OFD1 sequence downstream (exons 17–20) and upstream (exons 20–23) of the mutation site. Lower part: PCR bands of housekeeping gene GAPDH run in parallel. GADPH, glyceraldehyde 3-phosphate dehydrogenase; IDRs, intrinsically disordered regions; JBTS10, Joubert syndrome type 10; PCD, primary ciliary dyskinesia; RT-PCR, real-time PCR; SGBS2, Simpson-Golabi-Behmel syndrome type 2; WT, wild type.

The previously reported mutations of OFD1 cause three X-linked ciliopathies: a dominant, male-lethal oral-facial-digital syndrome type 1 (OFDS1) and two recessive diseases: Simpson-Golabi-Behmel syndrome type 2 (SGBS2) and Joubert syndrome type 10 (JBTS10).4 13 19 20 Dysmorphic features, skeletal abnormalities, neurological as well as renal symptoms are characteristic of these syndromes (see Supplementary Data for the description of the clinical symptoms). The majority of identified OFD1 mutations introduce premature termination codons (PTCs), yielding a protein that is truncated or weakly expressed due to nonsense-mediated decay (NMD).21 22 The severity of the phenotype (lethality, the intensity of the symptoms listed above) generally decreases for mutations localised closer to the C-terminus of OFD1 (figure 1A).21–23

Phenotypic manifestation of OFD1 mutations is highly variable between the different syndromes and among patients with the same syndrome,23–25 even within families carrying the same pathogenic variant.21 26 Nevertheless, the presence of brain defects and skeletal (digital, facial or dental) abnormalities is typical. On the contrary, respiratory symptoms in patients with OFD1 mutations have been limited to JBTS10 patients,21–23 27 a single family with SGBS2 (6) and only few OFDS1 cases25 28; moreover, such symptoms were reported as only supplementary to the predominant neurological or skeletal symptoms.

Here, we report on four novel protein-truncating mutations localised in exons 20 and 21 of the OFD1 gene, detected in patients with a clinical presentation of PCD (including recurrent respiratory infections, situs inversus and otitis media), in whom no severe neurological, renal or skeletal symptoms were observed. The mild phenotype of our patients suggests that, in case of variants affecting the C-terminal part of the protein, the spectrum of OFD1-related disorders may include ‘classical’ PCD without any syndromic features.


Biological material from patients

The full cohort of Slavic PCD patients consisted of 380 families of Polish and 17 of Slovak origin, who were classified as PCD in concordance with European guidelines in PCD.29 Classical clinical symptoms included neonatal respiratory distress, recurrent upper and lower respiratory tract infections, sinusitis, bronchiectasis, otitis media, and in some—abnormalities in left-right body asymmetry, mainly situs inversus. Where possible, clinical diagnosis was confirmed by any of the following criteria: (i) low nasal nitric oxide (nNO) production (<77 nL/min) measured using velum closure on a chemiluminescent nitric oxide analyzer; (ii) abnormal ciliary motion in high-speed videomicroscopy (HSVM); (iii) cilia ultrastructure defects in transmission electron microscopy (TEM) or immunofluorescence (IF). The details of cilia analysis are described in the Supplementary file.

Body mass and height were presented according to the percentile charts for growth and nutritional status for Polish children and adolescents.30 Head circumference was analysed according to Bushby et al.31 Written informed consent was obtained from PCD patients or their guardians and from family members and volunteers, by following protocols approved by the Ethics Review Board at the Medical University in Poznan (# 435/13).

Respiratory epithelial cells were obtained by nasal biopsy using a Cytobrush cell collector (Cooper Surgical) or during a routine diagnostic bronchoscopy. Brushing samples were suspended in RPMI medium, spread on microscope slides and examined by HSVM or dried at room temperature before staining. Brushing samples for TEM were immediately transferred to TEM fixation buffer and stored at 4°C until further processing. Procedures for nasal NO measurement, magnetic resonance brain imaging, genomic DNA isolation, OFD1 expression analysis as well as methods used for motile cilia analysis (TEM, HSVM and IF), are described in the Supplementary File.

Sequencing and genotyping of the patients and families with PCD

Whole-exome sequencing (WES) of the genomic DNA was performed for 120 unrelated PCD patients from the cohort. WES was performed at ×100 coverage by commercial providers (BGI, HongKong and Macrogen, Seoul), using SureSelectXT V5 PostCap exome library preparation system (Agilent Technologies, CA, USA) and the HiSeq4000 platform (Illumina, CA, USA). For the details of WES and deleterious variant identification, see the Supplementary File. Whenever parents or siblings of the patient were available, segregation of the PCD-causing variants in a family was examined by dideoxy sequencing of PCR-amplified relevant exons.

In the remaining PCD patients from the collection (in whom no causative mutation in other PCD genes was previously identified), the coding sequence of OFD1 was screened to estimate a possible involvement of OFD1 mutations. The samples were screened using single-stranded conformation polymorphism (SSCP) analysis and dideoxy sequencing, as described in Ziętkiewicz et al.32 The details on these analyses (primers, PCR and SSCP conditions) are available from the authors on request.

OFD1 sequence nomenclature

Mutations were described according to the nomenclature recommendations on the checklist for the description of sequence variants ( The coding sequence numbering was that of Ensembl (release 94) transcript OFD1-201 (ENST00000340096.10, RefSeq: NM_003611) and protein ENSP00000344314.6 (RefSeq: NP_003602). The newly identified OFD1 variants (p.Gln872fs*26, p.Tyr916fs*7, p.Glu933*X, p.Glu939*) were submitted to ClinVar (accession numbers: SCV000882676.1, SCV000882864.1; SCV000882865.1 and SCV000882866.1, respectively).


Patients genotyping

Genetic screening using whole-exome next generation sequencing was performed in 120 unrelated PCD individuals, in whom previous screening of over 80 exons in 13 genes most frequently involved in PCD pathogenesis in the Slavic population has not identified any deleterious mutations (for the details, see Supplementary Materials and Methods).

Among a number of previously undescribed variants, two novel truncating, hemizygous mutations in the OFD1 gene were detected in two unrelated male patients, 855 and 581. Patient 855 had a 5 bp frameshift deletion in exon 20 (c.2615-2619delAAATT, p.Gln872fs*26); patient 581 had a nonsense mutation in exon 21 (c.2797G>T, p.Glu933*). Extended screening of the whole coding sequence of OFD1, performed using SSCP analysis and dideoxy sequencing in the remaining part of our Slavic PCD cohort without mutations in known PCD genes, identified other deleterious variants in two unrelated males (343 and 961). Patient 343 carried a frameshift insertion in exon 20 (c.2746-2747insT; p.Tyr916fs*7), whereas patient 961 carried a nonsense mutation in exon 21 (c.2815G>T; p.Glu939*); no other deleterious changes were found in patients 343 and 961 screened for the whole range of mutations repetitively found in the Polish PCD cohort (data not shown). In total, four novel pathogenic variants in OFD1 were found: two in exon 20 (patients 855, 343) and two in exon 21 (patients 581, 961) (figure 1B).

The follow-up dideoxy sequencing of DNA from the available family members confirmed the recessive X-linked inheritance in patients 343 (family 213), 855 (family 384) and 961 (family 440), with pathogenic OFD1 variants present only in the non-symptomatic carrier mothers. In family 259, the pathogenic c.2797G>T variant (p.Glu993*) identified in patient 581 was not found in any of the parents, suggesting a de novo mutation (figure 1B; see Supplementary File for methodology). Analysis of the OFD1 sequence in DNA isolated from the patient’s nails confirmed the presence of the pathogenic variant in exon 21 in the tissue descending from the embryonic ectoderm. This indicated that the pathogenic allele in patient 581 resulted from a mutation in the maternal germ cell line, rather than in the developing embryo, thus excluding the possibility of a mosaic presence of the pathogenic variant in the patient. Analysis of cDNA in patient 581 revealed the presence of a prematurely truncated OFD1 transcript, consistent with the nonsense mutation in exon 21; no signs of NMD were observed (figure 1C).

Phenotypes of four patients with PCD with pathogenic OFD1 variants

The facial or digital defects, typical for OFDS1, SGBS2 or JBTS10, were unusually mild in our patients. They included only polydactyly in patient 961, mild facial dysmorphia in patient 343 and planovalgus feet and increased head circumference in patient 855. Mild learning difficulties were reported in patients 961 and 343; the latter also showed delayed eye–hand coordination. Intellectual disability was absent in patients 855 and 581 and MRI of the brain, available for these patients did not reveal the presence of molar tooth sign, MTS, a pathognomonic feature of JBS10 (online supplementary figure S1).

Supplemental material

The major phenotype in four male patients with truncating mutations localised in OFD1 exon 20 or 21 was typical for PCD and included early-in-life recurrent obstructive bronchitis, bronchiectasis, sinusitis, and otitis media with hearing loss; nNO levels available for two patients had borderline values for PCD (table 1; see online supplementary data for more details on patients’ clinical phenotypes). In one case (patient 855) situs inversus was found, suggesting that the functional impairment involved motile cilia both in the respiratory tract and in the embryonic node. To better characterise the effect of the mutations on the motile cilia function, a number of analyses were performed.

Table 1

Characteristics of PCD patients with the identified OFD1 mutations

HSVM analyses performed using respiratory epithelium obtained from patients 581 and 855 revealed stiff, unsynchronised cilia beating with reduced amplitude (black arrows) (online supplementary videos 1–4). In patient 855, cells with immotile or nearly immotile cilia were also observed (online supplementary video 1, white arrows); in another epithelium sample, a mix of normal cilia beating and stiff beating pattern was observed (online supplementary videos 5,6). In both patients, no signs of rotation were visible (online supplementary videos 5–8), but mucus was frequently observed.

Cilia in both patients, 855 and 581, appeared relatively sparse compared with a control sample; they also appeared much longer (compare online supplementary videos 5–8 with online supplementary videos 9,10). In patient 855, a number of cells with extremely long, unsynchronised cilia, entering and exiting the optical plane of the microscope, were observed (online supplementary videos 1, 3 and 5, black arrows).

Consistently with the HSVM results, immunostaining of the airway epithelium from patients 855 and 581, using antibodies against basal body and axonemal markers, revealed a significantly reduced proportion of ciliated cells: it was 2.2-fold to 5.9-fold times lower, respectively, compared with control samples (p<0.0001; figure 2C; method description in online supplementary data). Moreover, airway cilia length in the phase-contrast images was variable and on average significantly higher than in the controls (855: 9.69±5.47 µm; 581: 6.74±2.73 µm, control: 3.33±0.9 µm; p<0.0001) (figure 2D, online supplementary figure S2).

Figure 2

Respiratory cilia phenotype in patients with C-terminal OFD1 mutations. (A) Immunofluorescent staining of OFD1 and centrin in airway epithelial cells from patients 581 and 855 showing expression of the truncated OFD1 (red) and cytoplasmic accumulation of basal body marker, centrin (green). Nuclei were stained with DAPI, magnification ×100. (B.) Localisation of ciliary axoneme proteins GAS8 (red) or DNAI2 (green) in patient 855 indicating correct axonemal structure, but abnormal cilia length in the same cells. Nuclei were stained with DAPI, magnification ×100. (C) Analysis of the proportion of ciliated cells in patients 855 and 581 indicates its significant reduction compared with the control. Asterisks indicate p values <0.0001. (D) Cilia length analysis in patients 855 and 581 shows abnormally long cilia in both patients. Asterisks indicate p values <0.0001. (E) TEM cross section of airway cilia from patient 581 showing normal 9+2 structure of the axoneme. Scale bars are 200 nm. (F) TEM images of the airway epithelial cells from patient 581 showing sparse cilia, microvilli and cytoplasmic accumulation of the ciliary basal bodies with the characteristic triplets of peripheral microtubules (insets). Scale bars are 1 µm. DAPI, 4′,6-diamidino-2-phenylindole; TEM, transmission electron microscopy.

Re-examination of the archival TEM samples of the respiratory epithelium from patient 581 revealed cells mainly covered with microvilli, with only single cilia on the surface (figure 2F, online supplementary figure S3). Ciliary basal bodies were found primarily in the cytoplasm, with only few basal bodies present at the cells surface and elongated into cilia (n=64 and n=12, respectively, per 12 cells analysed). This was in contrast to a control sample, where numerous cilia elongated from basal bodies docked at the cell membrane (figure 2F, online supplementary figure S3); only a single basal body was found in the cytoplasm per eight control cells examined.

Immunofluorescent staining of the cells with an antibody against OFD1 revealed that the mutated protein was expressed in both patients, 581 and 855 (figure 2A, online supplementary figure S3). In control cells, OFD1 colocalised with centrin, the basal body marker. In patients 581 and 855, the strongest OFD1 and centrin signals were seen in the central/apical part of the cytoplasm, indicating that only a minority of basal bodies was anchored at the apical cellular membrane in the patients (figure 2A, online supplementary figure S3). Immunofluorescent staining using antibodies against GAS8, DNAI2 and DNAH5 was mostly cytoplasmic, consistent with the relatively low number of ciliated cells in the patient samples. However, when cilia were present in the patients, the axonemal markers staining suggested a normal architecture of the ciliary axoneme (figure 2B, online supplementary figure S3).

The normal ultrastructure of the axoneme was confirmed in TEM analysis of cilia from patient 581. The 9+2 pattern and the presence of dynein arms were observed in almost all the analysed cross sections (n=12) (figure 2E); only one cilium showed microtubular abnormalities, most probably due to secondary damage. The insufficient number of cilia cross sections in the patient (n=12) did not allow a quantitative analysis of the ciliary ultrastructure.


Clinical symptoms in patients with OFD1 mutations

OFD1 protein is a component of centrioles and ciliary basal bodies, essential for the biogenesis of primary and motile cilia.12 Defects in OFD1 lead to severe syndromic ciliopathies (OFDS1, SGBS2, JBST10), whose symptoms are caused by the dysfunction of organs or tissues expressing primary cilia (skeleton, nervous system, kidneys, retina), and rarely by the respiratory dysfunction due to motile cilia defects.

Here, we report four novel truncating mutations localised in exons 20 and 21 of OFD1 in unrelated PCD patients, whose clinical phenotype was typical for motile cilia defects, with a severe respiratory dysfunction in all four and presence of a situs inversus in one case. Limited contacts with patients did not allow us to apply a full diagnostic path to all the cases, but the scores using PICADAR, a predictive tool used for PCD diagnostics,33 indicated that the patients were properly identified as having PCD. Relatively mild symptoms related to primary cilia defects (subtle neural or skeletal symptoms, lack of renal, retinal or cardiac defects) ruled out a diagnosis of OFDS1 or SGBS2 in all four patients. No intellectual disability was reported in patients 855 and 518. While the latter did not present even mild skeletal abnormalities, the increased head circumference and planovalgus feet in patient 855 could have suggested this patient’s classification as JBTS10. However, MTS, a pathognomonic feature of all types of Joubert syndrome-related disorders,19 was absent in the magnetic resonance mid-brain images of 581 and 855, excluding JBTS10 diagnosis in both these patients. On the other hand, a combination of mild intellectual disability and polydactyly observed in patients 961 and patient 343 suggested mild JBTS10 with severe respiratory symptoms and no renal or cardiac involvement; mid-brain imaging in these patients would be necessary to confirm this diagnosis.

Analysis of other clinical symptoms observed in our patients also pointed to PCD or JBS10, rather than to OFDS1 or SGBS2. Situs inversus in patient 855 is more indicative of PCD, where about 50% of affected patients present laterality defects27 28 34; situs inversus has been also reported (although rarely) in JBTS10 cases, but not in SGBS2 or OFDS1 patients.4 24 35 Conductive hearing loss due to recurrent ear infections, present in all our patients, is relatively frequent in PCD patients.1 33 Not found in SGBS2,4 it has been reported in 6% of OFDS1 patients (; its presence in 27% of Dutch Joubert syndrome patients has been related to pathogenic variants in various genes.36 Obesity and hyperphagia, although previously described in Joubert syndrome-related disorders caused by mutations in other genes,19 37 38 as well as other in cilia-related syndromes, such as Bardet-Biedl syndrome39, have not been reported in OFDS1, SGBS2 or JBTS10.

Recurrent airway infections, bronchiectasis, otitis media, and sinusitis, have been occasionally observed in patients with syndromic disorders caused by mutations in the OFD1 gene.4 21 22 27 28 40–42 For the majority of OFDS1 cases, with mutations introducing PTC between exons 1 and 17 of OFD1, respiratory symptoms have not been reported. This may have been caused by the lethality of male embryos and the prevailing, severe neurological and dysmorphic features in females. However, in few live-born OFDS1 patients with PTC mutations in exon 16 (at amino acid positions 608, 630 or 728), in spite of the exhaustive clinical phenotype description available, no reference to any respiratory involvement has been mentioned.21 41 42

So far, only three mutations in OFDS1 patients, in exons 13, 17 and at a donor splice site in intron 17, have been explicitly reported to cause respiratory symptoms25 28 (figure 3). Almost all other pathogenic OFD1 variants, for which respiratory symptoms have been reported, were in JBTS10 or SGBS2 cases (figure 3). All these patients had PTC mutations localised in the sequence encoding the central and/or C-terminal part of the protein: one in exon 16 caused SGBS2; five, in exons 20 and 21, were found in JBTS10 patients (see figure 3).4 21–23 27 28 41 42 However, respiratory symptoms in all SGBS2 or JBTS10 patients have been consistently reported to coexist with a spectrum of severe syndromic abnormalities (neurological/skeletal/renal); in JBTS10 cases, they were always accompanied by the presence of the pathognomonic MTS in the brain.23 27 Two patients with OFD1 mutations in exon 21 have been reported before to have clinical symptoms consistent with PCD.43 In one of the patients (with OFD1 mutation c.2789_2793del5 (p.Ile930fs*8)), previously reported in JBTS10,22 43 PCD symptoms accompanied intellectual delay and polydactyly.43 The other patient, with the previously undescribed OFD1 mutation (c.2868delT (p.Pro957fs*2)), presented PCD symptoms without intellectual delay or polydactyly, similar to our patients 581 and 855. Unfortunately, no data cilia defects at the cellular level in these patients were provided, making any comparison with our observations not possible.

Figure 3

Pathogenic OFD1 variants in exons 16–23 occurring with respiratory symptoms. Alternating OFD1 exons are shown in white or grey; exon 23 contains coding sequence (white) and a long 3′ UTR (light grey). Bars under the exons indicate different structural domains (red—coiled coils, according to (N coils); green—disordered regions (IDRs, according to (MobiDB Lite)). Upper part: exonic localisation of the pathogenic protein variants (for the clarity, aminoacids are shown using a one letter IUPAC (International Union of Pure and Applied Chemistry) code). Colour of the variant indicates the major associated phenotype (dark red: OFDS1; green: SGBS2; dark blue: JBTS10; light blue: PCD-like symptoms or mutations reported in this manuscript). Variants associated with respiratory symptoms are shown in bold italic; superscripts in the variant’s name indicate references: B,4 C,23 D,41 H,43 M,27 R,42 T,40 TR1,21 TR2,22 Tsu,28 new (this study). IDRs, intrinsically disordered regions.

Our study confirms that OFD1 truncations localised in exons 20 and 21 of OFD1 can lead to a respiratory phenotype not necessarily associated with severe neurological/skeletal/renal symptoms. This extends the spectrum of OFD1-related disorders to a ‘classical’ PCD without obvious syndromic features, and indicates that exons 20 and 21 of the OFD1 gene should be included in a regular mutation screening of PCD patients. It also suggests that truncating mutations localised near the C-terminus of the OFD1 protein, while severely impairing ciliary motility (also in the embryonic node), may have only a limited effect on primary cilia function.

The impact of newly identified OFD1 mutations on the ciliary phenotype

No defects of the axonemal ultrastructure were observed in TEM or IF analysis of cilia from our patients. However, the reduced number of cilia in the airway epithelium cells was consistently observed in HSVM and IF. TEM analysis confirmed that this deficiency was related to the defective cilia anchoring; cilia grown from the sparse basal bodies docked at the apical membrane were characterised by the abnormal length. While the reduced number of cilia may not be a direct result of the cytoplasmic accumulation of basal bodies, these observations are in agreement with the earlier studies in animal models, which suggested that OFD1 mutations may cause defective anchoring of motile cilia, and a variable length of motile cilia and ciliary basal bodies/centrioles.10 11

The restricted access to patients’ biological material limited the ability to perform in vitro culture of the patients’ airway epithelium, which would confirm the primary character of cilia/basal body defects. Future studies using genetically modified animal or cellular models with different OFD1 mutations will allow filling this knowledge gap.

Taking into account the motile cilia sparsity in our patients, it is possible that C-terminal OFD1 mutations cause phenotypic defects, which result in reduced generation of motile cilia (RGMC).44 45 RGMC phenotype has been previously observed in PCD patients with mutations in CCNO or MCIDAS genes, encoding proteins that act at the very early stages of motile cilia biogenesis.44 45 Compared with these proteins, OFD1 plays a role in the relatively late stage, that is, docking of ciliary basal bodies.12 22 OFD1 mutations can therefore reduce the number of properly anchored motile cilia and promote the accumulation of basal bodies in the cytoplasm, as evidenced by the studies in model organisms10 12 and humans [22 28 and this study].

The correlation between phenotype severity and the site of OFD1 mutations

Severity of symptoms in disorders related to OFD1 mutations that introduce PTC codons is inversely proportional to the length of the coding sequence upstream from the mutation.21 23 46 The male-lethal, dominant OFDS1 cases are related to PTCs localised in exons 1–17,28 while mutations in exons 17–23 cause recessive disorders with varying severity: SGBS2, JBTS104 20 or even PCD, as suggested by our study and the others.43 PTC mutations may lead to NMD, resulting in the lack or a severely reduced level of the protein.28 47 If NMD is not present, protein truncations of different extent may lead to the loss of different functional OFD1 domains (LisH motif or CC domains), thus changing binding affinity of OFD1 to some of its multiple interactors.11 23 46 48 Any of these scenarios may lead to the impairment of OFD1-related processes at cilia or centrosomes. Indeed, PTC mutations at amino acids Asn709 (exon 16), Glu923 and Lys948 (exon 21) have been shown to significantly affect OFD1 binding to SDCCAG8, the centrosomal protein,48 and lebercillin, the primary cilia protein23; they also affected the cellular localisation of OFD1.23 48

The majority of OFD1 mutations previously reported in patients with the respiratory symptoms (figure 3) are localised downstream from exon 16. In the absence of NMD (as demonstrated in patient 581), they may lead to truncation of the C-terminal part of the protein. This region includes the CC7 domain (amino acid positions 868–925, Ensembl release 94), potentially important for protein–protein interactions. However, more than half of the 13 ‘respiratory’ PTC mutations, being localised downstream to CC7, may not directly affect this domain in the absence of NMD (figure 3). In contrast, all the ‘respiratory’ PTCs would result in the loss of the IDR encompassing amino acids 963–991 (figure 3). As even small changes in IDRs can significantly affect protein binding kinetics,19 these PTCs could result in suboptimal binding of some protein partners interacting with the C-terminal part of OFD1 and important for biogenesis of motile cilia. However, the same interactions would also be disturbed as a result of the upstream PTC mutations found in OFDS1 patients, in whom motile cilia dysfunction has not been observed. This, perhaps, can be explained by the mode of inheritance. In contrast to SGB2, JBTS10 and PCD, OFDS1 is male-lethal and dominant in females; a single copy of the wild-type allele in female patients, which cannot sustain the proper functioning of primary cilia, may be sufficient to prevent the manifestation of the motile cilia phenotype.

Finally, considering a multitude of the already identified OFD1 roles related to centrosomes, ciliogenesis, translation and histone acetylation,11 28 49 50 it is possible that truncating mutations located downstream from the OFD1 exon 16 affect other, yet unidentified OFD1 functions.


The authors gratefully acknowledge technical support by Katarzyna Voelkel and Ewa Rutkiewicz, TEM data provision by Dr Anna Sulikowska-Rowińska, TEM ultrastructure analysis by Regan Doherty and Dr Anton Page, bioinformatics support by Dr Roman Jaksik and critical comments by Dr Patrycja Daca-Roszak. Clinical support of Professor Jaroslaw Szydlowski, Department of Paediatric Otolaryngology, Poznan University of Medical Sciences in Poznan is gratefully acknowledged.



  • AR and MD contributed equally.

  • Contributors EZ, ZBB and AR participated in the design of the study. ZBB, AR, AW, MD, AP, KG, HG, JS collected and/or generated the data. ZBB, AR, MD, EZ, AP, HD, KG analysed and/or interpreted the data. ZBB, EZ, AR, MW drafted the manuscript. All authors corrected the manuscript.

  • Funding The authors of this manuscript have been supported by the funding from the Polish National Science Center, Poland, granted based on decision number DEC 2014/13/B/NZ2/03858 (EZ) and DEC- 2013/09/D/NZ4/01692 (ZBB). The funding bodies did not have any influence on the design of the study, or its results or the manuscript preparation. Authors of this manuscript are participants in BEAT-PCD project (COST Action BM 1407).

  • Competing interests ZBB, AR, MD, AW, MW and EZ report grants from National Science Center, Poland, during the conduct of the study (funding granted to EZ and ZBB). ZBB reports also personal fees from National Science Centre, Poland during the conduct of the study. ZBB and AW report personal fees from National Science Centre, outside the submitted work.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.