Pathogenesis Of Primary Ciliary Dyskinesia Research Articles

Identification of a novel RPGR mutation associated with retinitis pigmentosa and primary ciliary dyskinesia in a Slovak family: a case report

BackgroundThe mutations in the RPGR (retinitis pigmentosa GTPase regulator) gene are the most common cause of X-linked retinitis pigmentosa (XLRP), a rare genetic disorder affecting the photoreceptor cells in the retina. Several reported cases identified this gene as a genetic link between retinitis pigmentosa (RP) and primary ciliary dyskinesia (PCD), characterised by impaired ciliary function predominantly in the respiratory tract. Since different mutations in the same gene can result in various clinical manifestations, it is important to describe a correlation between the gene variant and the observed phenotype.MethodsTwo young brothers from a non-consanguineous Slovak family with diagnosed retinal dystrophy and recurrent respiratory infections were examined. Suspected PCD was diagnosed based on a PICADAR questionnaire, nasal nitric oxide analysis, transmission electron microscopy, high-speed video microscopy analysis, and genetic testing.ResultsWe identified a novel frameshift RPGR mutation NM_001034853: c.309_310insA, p.Glu104Argfs*12, resulting in a complex X-linked phenotype combining PCD and RP. In our patients, this mutation was associated with normal ultrastructure of respiratory cilia, reduced ciliary epithelium, more aciliary respiratory epithelium, shorter cilia, and uncoordinated beating with a frequency at a lower limit of normal beating, explaining the clinical manifestation of PCD in our patients.ConclusionThe identified novel pathogenic mutation in the RPGR gene expands the spectrum of genetic variants associated with the X-linked PCD phenotype overlapping with RP, highlighting the diversity of mutations contributing to the disorder. The described genotype–phenotype correlation can be useful in clinical practice to recognise a broader spectrum of PCD phenotypes as well as for future research focused on the genetic basis of PCD, gene interactions, the pathways implicated in PCD pathogenesis, and the role of RPGR protein for the proper functioning of cilia in various tissues throughout the body.

Biallelic Variants in CCDC39 Gene Lead to Primary Ciliary Dyskinesia and Kartagener Syndrome.

Background Primary ciliary dyskinesia (PCD) is a clinical syndrome characterized by cilia with an abnormal structure or function. Its main clinical manifestations comprise chronic bronchitis, cough, recurrent respiratory infections, situs inversus, and male infertility. Single-gene variants are widely assumed to be the main cause of this rare disease, and more than 40 genes have been described to be associated with its onset. CCDC39 is essential for assembling the inner dynein arms and dynein regulatory complex and is important in cilia motility. CCDC39 variants were reported as a monogenic etiology of PCD. Methods This study investigated two unrelated Chinese patients diagnosed as PCD. The chest computed tomography scan was performed to identify PCD phenotypes of the two probands. Considering the effect of PCD on male fertility, routine semen analysis, sperm morphology examination, and scanning electron microscopy were performed to assess the semen characteristics of male proband in family 2 (F2 II-1), who had a history of infertility. Subsequently, the peripheral blood samples of probands were collected to perform whole-exome sequencing (WES) to explore the possible genetic causes of this disease. Results Whole-exome sequencing revealed a homozygous CCDC39 variant in the female proband of family 1 (F1 II-1: c.286C>T:p.Arg96Ter) and two compound heterozygous CCDC39 variants in the male proband of family 2 (F2 II-1: c.732_733del: p.Ala245PhefsTer18; c.2800_2802dup:p.Val934dup). The two probands showed the typical PCD phenotypes, including chronic bronchitis, recurrent respiratory infections, and situs inversus. The male proband also showed oligoasthenoteratospermia with multiple morphological abnormalities of the sperm flagella. Additionally, CCDC39 protein level was significantly lower in the sperm of male proband than in the sperm from normal controls. Conclusion We identified a homozygous variant reported previously and two compound heterozygous variants of CCDC39 possibly responsible for PCD pathogenesis, expanding the variant spectrum of Chinese PCD, Kartagener syndrome, and morphological abnormalities of the sperm flagella involving CCDC39.

In vitro differentiation of ciliated cells in ALI-cultured human airway epithelium – The framework for functional studies on airway differentiation in ciliopathies

Primary cultures of the human airway epithelium (AE) cells are an indispensable tool in studies of pathophysiology of genetic and environmental pulmonary diseases, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and chronic obstructive pulmonary disease (COPD). Air-liquid interface (ALI) culture is the best method to follow the differentiation of ciliated cells, whose dysfunction forms the basis of PCD. Here, we used custom-designed Taqman Low Density Array (TLDA), qRT-PCR-based assay, to analyze expression of 14 AE genes in cells from healthy donors, cultured in ALI settings using Pneumacult medium, with the focus on genes involved in cilia differentiation and in PCD pathogenesis. The results of TLDA assay were compared with the bulk RNAseq analysis, and placed in the cellular context using immunofluorescent staining (IF) of ALI cultured cells.Expression analysis revealed culture time-related upregulation of the majority of cilia-related genes, followed by the appearance of respective protein signals visualized by IF. Strong correlation of TLDA with RNAseq results indicated that TLDA assay is a reliable and scalable approach to analyze expression of selected genes specific for different AE cell types. Characterization of temporal and inter-donor changes in the expression of these genes, performed in healthy donors and in well-defined ALI/Pnemacult culture conditions, provides a useful reference relevant for a broad spectrum of functional studies where the in vitro AE differentiation is in focus.

A novel CCDC39 mutation causes multiple morphological abnormalities of the flagella in a primary ciliary dyskinesia patient

Research questionMale infertility is a widespread symptom in patients with primary ciliary dyskinesia (PCD). PCD-related male infertility is often caused by asthenozoospermia, with barely normal sperm morphology. Multiple morphological abnormalities of the sperm flagella (MMAF) are a major cause of asthenozoospermia, characterized by various malformed morphologies of sperm flagella. To date, a limited number of genes have been suggested to be involved in the pathogenesis of both PCD and MMAF. What other genes associated with both PCD and MMAF are waiting to be discovered? DesignWhole-exome sequencing (WES) was performed to identify the pathogenic mutation associated with MMAF in a PCD patient. Peripheral venous blood and semen samples were collected from the PCD patient. Transmission electron microscopy (TEM), immunofluorescence staining and western blotting were conducted to confirm the pathogenicity of the identified mutation. ResultsA novel homozygous mutation in CCDC39, c.983 T>C (p. Leu328Pro), was identified in two PCD-affected siblings of a consanguineous family showing a typical PCD phenotype, while the proband was infertile, which is associated with characterized MMAF. Furthermore, TEM revealed the abnormal ultrastructure of the patient's sperm flagella. Moreover, immunofluorescence staining revealed that CCDC39 was almost undetectable in the spermatozoa, which was further confirmed by western blotting. The outcome of intracytoplasmic sperm injection (ICSI) in the patient with the CCDC39 mutation was also favourable. ConclusionThis study demonstrates that a novel loss-of-function mutation of CCDC39 is involved in the pathogenesis of PCD and MMAF and initially reported that ICSI treatment has a good outcome. Therefore, the novel variant of CCDC39 contributes to the genetic diagnosis, counselling and treatment of male infertility in PCD patients with MMAF phenotype.

Primary ciliary dyskinesia relative protein ZMYND10 is involved in regulating ciliary function and intraflagellar transport in Paramecium tetraurelia

Cilia are highly conserved in most eukaryotes and are regarded as an important organelle for motility and sensation in various species. Cilia are microscopic, hair-like cytoskeletal structures that protrude from the cell surface. The major focus in studies of cilia has been concentrated on the ciliary dysfunction in vertebrates that causes multisymptomatic diseases, which together are referred to as ciliopathies. To date, the understanding of ciliopathies has largely depended on the study of ciliary structure and function in different animal models. Zinc finger MYND-type containing 10 (ZMYND10) is a ciliary protein that was recently found to be mutated in patients with primary ciliary dyskinesia (PCD). In Paramecium tetraurelia, we identified two ZMYND10 genes, arising from a whole-genome duplication. Using RNAi, we found that the depletion of ZMYND10 in P. tetraurelia causes severe ciliary defects, thus provoking swimming dysfunction and lethality. Moreover, we found that the absence of ZMYND10 caused the abnormal localization of the intraflagellar transport (IFT) protein IFT43 along cilia. These results suggest that ZMYND10 is involved in the regulation of ciliary function and IFT, which may contribute to the study of PCD pathogenesis.

Motile cilia genetics and cell biology: big results from little mice.

Our understanding of motile cilia and their role in disease has increased tremendously over the last two decades, with critical information and insight coming from the analysis of mouse models. Motile cilia form on specific epithelial cell types and typically beat in a coordinated, whip-like manner to facilitate the flow and clearance of fluids along the cell surface. Defects in formation and function of motile cilia result in primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder with a well-characterized phenotype but no effective treatment. A number of model systems, ranging from unicellular eukaryotes to mammals, have provided information about the genetics, biochemistry, and structure of motile cilia. However, with remarkable resources available for genetic manipulation and developmental, pathological, and physiological analysis of phenotype, the mouse has risen to the forefront of understanding mammalian motile cilia and modeling PCD. This is evidenced by a large number of relevant mouse lines and an extensive body of genetic and phenotypic data. More recently, application of innovative cell biological techniques to these models has enabled substantial advancement in elucidating the molecular and cellular mechanisms underlying the biogenesis and function of mammalian motile cilia. In this article, we will review genetic and cell biological studies of motile cilia in mouse models and their contributions to our understanding of motile cilia and PCD pathogenesis.

Ultrastructural Sperm Flagellum Defects in a Patient With CCDC39 Compound Heterozygous Mutations and Primary Ciliary Dyskinesia/Situs Viscerum Inversus.

Introduction: Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disease characterized by structural or functional motile cilia abnormalities. Up to 40 different genes seem, at the moment, to be involved in the pathogenesis of PCD. A number of ultrastructural defects have also been reported in sperm flagella, but the sperm mitochondrial membrane potential (MMP) has never been described in these cases.Aim: The aim of this study was to report the sperm MMP and ultrastructural abnormalities of the sperm flagella found in a patient with PCD and situs inversus (Kartagener syndrome) and its characterization from the genetic point of view.Methods: Transmission electronic microscopy (TEM) analysis was used to evaluate flagella ultrastructure. The genetic testing was performed by next-generation sequencing. Sperm DNA fragmentation and MMP were also evaluated by flow cytometry.Results: We report here the case of an 18-year-old male patient with PCD and situs inversus and severe oligo-astheno-teratozoospermia. TEM analysis of his spermatozoa showed an abnormal connecting piece. The mid piece appeared abnormally thickened, with cytoplasmic residue, dysplasia of fibrous sheath, loss of the outer dynein arms (ODAs), truncated inner dynein arms, and supernumerary outer fibers. The percentage of spermatozoa with fragmented DNA was normal, whereas a high percentage of spermatozoa had low MMP, suggesting an altered mitochondrial function. The genetic analysis showed the presence of c.610-2A > G, p.Arg811Cys compound heterozygous mutations in the CCDC39 gene.Conclusion: The case herein reported suggests that the high percentage of sperm with low MMP may play a role in the pathogenesis of asthenozoospermia in patients with Kartagener syndrome. In addition, we report, for the first time, the missense variant p.Arg811Cys in the CCDC39 gene in a patient with Kartagener syndrome. Although in silico analysis predicts its damaging potential, its clinical meaning remains unclear.

Induced Pluripotent Stem Cells for Primary Ciliary Dyskinesia Modeling and Personalized Medicine.

Primary ciliary dyskinesia (PCD) is a rare and heterogeneous genetic disorder that affects the structure and function of motile cilia. In the airway epithelium, impaired ciliary motion results in reduced or absent mucociliary clearance that leads to the appearance of chronic airway infection, sinusitis, and bronchiectasis. Currently, there is no effective treatment for PCD, and research is limited by the lack of convenient models to study this disease and investigate innovative therapies. Furthermore, the high heterogeneity of PCD genotypes is likely to hinder the development of a single therapy for all patients. The generation of patient-derived, induced pluripotent stem cells, and their differentiation into airway epithelium, as well as genome editing technologies, could represent major tools for in vitro PCD modeling and for developing personalized therapies. Here, we review PCD pathogenesis and then discuss how human induced pluripotent stem cells could be used to model this disease for the development of innovative, patient-specific biotherapies.

Mutations in C11orf70 Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry Due to Defects of Outer and Inner Dynein Arms

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling.

Neutrophils from Patients with Primary Ciliary Dyskinesia Display Reduced Chemotaxis to CXCR2 Ligands

Primary ciliary dyskinesia (PCD), cystic fibrosis (CF), and chronic obstructive airway disease are characterized by neutrophilic inflammation in the lungs. In CF and chronic obstructive airway disease, improper functioning of neutrophils has been demonstrated. We hypothesized that the pulmonary damage in PCD might be aggravated by abnormal functioning neutrophils either as a primary consequence of the PCD mutation or secondary to chronic inflammation. We analyzed chemotactic responses and chemoattractant receptor expression profiles of peripheral blood neutrophils from 36 patients with PCD, 21 healthy children and 19 healthy adults. We stimulated peripheral blood monocytes from patients and healthy controls and measured CXCL8 and IL-1β production with ELISA. PCD neutrophils displayed reduced migration toward CXCR2 ligands (CXCL5 and CXCL8) in the shape change, microchamber and microslide chemotaxis assays, whereas leukotriene B4 and complement component 5a chemotactic responses were not significantly different. The reduced response to CXCL8 was observed in all subgroups of patients with PCD (displaying either normal ultrastructure, dynein abnormalities or central pair deficiencies) and correlated with lung function. CXCR2 was downregulated in about 65% of the PCD patients, suggestive for additional mechanisms causing CXCR2 impairment. After treatment with the TLR ligands lipopolysaccharide and peptidoglycan, PCD monocytes produced more CXCL8 and IL-1β compared to controls. Moreover, PCD monocytes also responded stronger to IL-1β stimulation in terms of CXCL8 production. In conclusion, we revealed a potential link between CXCR2 and its ligand CXCL8 and the pathogenesis of PCD.

Aminoglycoside-stimulated readthrough of premature termination codons in selected genes involved in primary ciliary dyskinesia

ABSTRACTTranslational readthrough of premature termination codons (PTCs) induced by pharmacological compounds has proven to be an effective way of restoring functional protein expression and reducing symptoms in several genetic disorders. We tested the potential of different concentrations of several aminoglycosides (AAGs) for promoting PTC-readthrough in 5 genes involved in the pathogenesis of primary ciliary dyskinesia, an inherited disorder caused by the dysfunction of motile cilia and flagella. The efficiency of readthrough stimulation of PTCs cloned in dual reporter vectors was examined in 2 experimental settings: in vitro (transcription/translation system) and ex vivo (transiently transfected epithelial cell line). PTC-readthrough was observed in 5 of the 16 mutations analyzed. UGA codons were more susceptible to AAG-stimulated readthrough than UAG; no suppression of UAA was observed. The efficiency of PTC-readthrough in vitro (from less than 1% to ∼28% of the translation from the corresponding wild-type constructs) differed with the AAG type and concentration, and depended on the combination of AAG and PTC, indicating that each PTC has to be individually tested with a range of stimulating compounds. The maximal values of PTC suppression observed in the ex vivo experiments were, depending on AAG used, 3–5 times lower than the corresponding values in vitro, despite using AAG concentrations that were 2 orders of magnitude higher. This indicates that, while the in vitro system is sufficient to examine the readthrough-susceptibility of PTCs, it is not sufficient to test the compounds potential to stimulate PTC-readthrough in the living cells. Most of the tested compounds (except for G418) at their highest concentrations did not disturb ciliogenesis in the cultures of primary respiratory epithelial cells from healthy donors.

The evolving spectrum of ciliopathies and respiratory disease.

Research on the biology of cilia, complex hair-like cellular organelles, has greatly informed our understanding of its crucial role in respiratory health and the pathogenesis of primary ciliary dyskinesia (PCD), including the genetics behind this condition. This review will summarize the current state of the art in the field highlighting its clinical implications. The genetics of PCD have exploded over the past few years as knowledge acquired from model systems has permitted the identification of genes that are key components of the ciliary apparatus and its function. In addition, clinical criteria and diagnostic tools have emerged that permit more clear identification of affected individuals. The rate of progress in the field continues to accelerate through international collaborative efforts and standardization of methods. Although the genetics behind PCD are complex, given the large number of genes associated with disease, as well as the large number of possible mutations even at the individual gene level, this knowledge is rapidly translating in improved diagnostics and hopefully in the near future in the identification of potential therapeutics.

ZMYND10--Mutation Analysis in Slavic Patients with Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a rare recessive disease with a prevalence of 1/10,000; its symptoms are caused by a kinetic dysfunction of motile cilia in the respiratory epithelium, flagella in spermatozoids, and primary cilia in the embryonic node. PCD is genetically heterogeneous: genotyping the already known PCD-related genes explains the genetic basis in 60–65% of the cases, depending on the population. While identification of new genes involved in PCD pathogenesis remains crucial, the search for new, population-specific mutations causative for PCD is equally important. The Slavs remain far less characterized in this respect compared to West European populations, which significantly limits diagnostic capability. The main goal of this study was to characterize the profile of causative genetic defects in one of the PCD-causing genes, ZMYND10, in the cohort of PCD patients of Slavic origin. The study was carried out using biological material from 172 unrelated PCD individuals of Polish origin, with no causative mutation found in nine major PCD genes. While none of the previously described mutations was found using the HRM-based screening, a novel frameshift mutation (c.367delC) in ZMYND10, unique for Slavic PCD population, was found in homozygous state in two unrelated PCD patients. Immunofluorescence analysis confirmed the absence of outer and inner dynein arms from the ciliary axoneme, consistent with the already published ZMYND10-mutated phenotype; cDNA analysis revealed the lack of ZMYND10 mRNA, indicating nonsense-mediated decay of the truncated transcript.

Primary ciliary dyskinesia

<h3>Educational aims</h3> To describe the emerging genetics of primary ciliary dyskinesia (PCD) and the heterogeneity of the disease To highlight the clinical symptoms and signs suggestive of PCD that should lead to consideration of diagnostic testing To highlight the difficulties in diagnosing PCD emphasising the need for specialist diagnostic centres To discuss current treatment strategies and highlight the lack of an evidence base for these <h3>Summary</h3> Primary ciliary dyskinesia (PCD) is a rare heterogeneous genetic disorder affecting ciliary function. Genes coding for various ciliary structural proteins or cytoplasmic proteins responsible for the assembly of cilia can be mutated resulting in abnormal ciliary function. However, despite the diversity of genotypes that can cause PCD the clinical phenotypes of PCD are all remarkably similar. The main clinical symptoms are caused by a lack of mucociliary clearance. Worryingly many patients are diagnosed late despite their classical, lifelong symptoms of a daily wet sounding cough and rhinosinusitis. Even when PCD is suspected, poor access to specialist diagnostic centres may delay diagnosis. Currently, diagnostic testing includes screening of nasal nitric oxide, followed by nasal brushing to obtain ciliated epithelial strips for high-speed video analysis of ciliary function. This is typically followed by transmission electron microscopy and in difficult cases by ciliated cell culture. Emerging tests including immunofluorescence and genetic examination are the focus of intense research and are likely to feature more in the future. Despite a greater understanding of the pathogenesis of PCD and improved diagnostic testing, management strategies are currently based on expert opinion with little, if any, evidence base.

The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans

Primary ciliary dyskinesia (PCD) is characterized by dysfunction of respiratory cilia and sperm flagella and random determination of visceral asymmetry. Here, we identify the DRC1 subunit of the nexin-dynein regulatory complex (N-DRC), an axonemal structure critical for the regulation of dynein motors, and show that mutations in the gene encoding DRC1, CCDC164, are involved in PCD pathogenesis. Loss-of-function mutations disrupting DRC1 result in severe defects in assembly of the N-DRC structure and defective ciliary movement in Chlamydomonas reinhardtii and humans. Our results highlight a role for N-DRC integrity in regulating ciliary beating and provide the first direct evidence that mutations in DRC genes cause human disease.

Loss-of-Function Mutations in LRRC6, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms, Cause Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a group of autosomal-recessive disorders resulting from cilia and sperm-flagella defects, which lead to respiratory infections and male infertility. Most implicated genes encode structural proteins that participate in the composition of axonemal components, such as dynein arms (DAs), that are essential for ciliary and flagellar movements; they explain the pathology in fewer than half of the affected individuals. We undertook this study to further understand the pathogenesis of PCD due to the absence of both DAs. We identified, via homozygosity mapping, an early frameshift in LRRC6, a gene that encodes a leucine-rich-repeat (LRR)-containing protein. Subsequent analyses of this gene mainly expressed in testis and respiratory cells identified biallelic mutations in several independent individuals. The situs inversus observed in two of them supports a key role for LRRC6 in embryonic nodal cilia. Study of native LRRC6 in airway epithelial cells revealed that it localizes to the cytoplasm and within cilia, whereas it is absent from cells with loss-of-function mutations, in which DA protein markers are also missing. These results are consistent with the transmission-electron-microscopy data showing the absence of both DAs in cilia or flagella from individuals with LRRC6 mutations. In spite of structural and functional similarities between LRRC6 and DNAAF1, another LRR-containing protein involved in the same PCD phenotype, the two proteins are not redundant. The evolutionarily conserved LRRC6, therefore, emerges as an additional player in DA assembly, a process that is essential for proper axoneme building and that appears to be much more complex than was previously thought.

Mutations in Radial Spoke Head Genes and Ultrastructural Cilia Defects in East-European Cohort of Primary Ciliary Dyskinesia Patients

Primary ciliary dyskinesia (PCD) is a rare (1/20,000), multisystem disease with a complex phenotype caused by the impaired motility of cilia/flagella, usually related to ultrastructural defects of these organelles. Mutations in genes encoding radial spoke head (RSPH) proteins, elements of the ciliary ultrastructure, have been recently described. However, the relative involvement of RSPH genes in PCD pathogenesis remained unknown, due to a small number of PCD families examined for mutations in these genes. The purpose of this study was to estimate the involvement of RSPH4A and RSPH9 in PCD pathogenesis among East Europeans (West Slavs), and to shed more light on ultrastructural ciliary defects caused by mutations in these genes. The coding sequences of RSPH4A and RSPH9 were screened in PCD patients from 184 families, using single strand conformational polymorphism analysis and sequencing. Two previously described (Q109X; R490X) and two new RSPH4A mutations (W356X; IVS3_2–5del), in/around exons 1 and 3, were identified; no mutations were found in RSPH9. We estimate that mutations in RSPH4A, but not in RSPH9, are responsible for 2–3% of cases in the East European PCD population (4% in PCD families without situs inversus; 11% in families preselected for microtubular defects). Analysis of the SNP-haplotype background provided insight into the ancestry of repetitively found mutations (Q109X; R490X; IVS3_2–5del), but further studies involving other PCD cohorts are required to elucidate whether these mutations are specific for Slavic people or spread among other European populations. Ultrastructural defects associated with the mutations were analyzed in the transmission electron microscope images; almost half of the ciliary cross-sections examined in patients with RSPH4A mutations had the microtubule transposition phenotype (9+0 and 8+1 pattern). While microtubule transposition was a prevalent ultrastructural defect in cilia from patients with RSPH4A mutations, similar defects were also observed in PCD patients with mutations in other genes.

Population specificity of the DNAI1 gene mutation spectrum in primary ciliary dyskinesia (PCD)

BackgroundMutations in the DNAI1 gene, encoding a component of outer dynein arms of the ciliary apparatus, are the second most important genetic cause of primary ciliary dyskinesia (PCD), the genetically heterogeneous recessive disorder with the prevalence of ~1/20,000. The estimates of the DNAI1 involvement in PCD pathogenesis differ among the reported studies, ranging from 4% to 10%.MethodsThe coding sequence of DNAI1 was screened (SSCP analysis and direct sequencing) in a group of PCD patients (157 families, 185 affected individuals), the first ever studied large cohort of PCD patients of Slavic origin (mostly Polish); multiplex ligation-dependent probe amplification (MLPA) analysis was performed in a subset of ~80 families.ResultsThree previously reported mutations (IVS1+2-3insT, L513P and A538T) and two novel missense substitutions (C388Y and G515S) were identified in 12 families (i.e. ~8% of non-related Polish PCD patients). The structure of background SNP haplotypes indicated common origin of each of the two most frequent mutations, IVS1+2-3insT and A538T. MLPA analysis did not reveal any significant differences between patients and control samples. The Polish cohort was compared with all the previously studied PCD groups (a total of 487 families): IVS1+2-3insT remained the most prevalent pathogenetic change in DNAI1 (54% of the mutations identified worldwide), and the increased global prevalence of A538T (14%) was due to the contribution of the Polish cohort.ConclusionsThe worldwide involvement of DNAI1 mutations in PCD pathogenesis in families not preselected for ODA defects ranges from 7 to 10%; this global estimate as well as the mutation profile differs in specific populations. Analysis of the background SNP haplotypes suggests that the increased frequency of chromosomes carrying A538T mutations in Polish patients may reflects local (Polish or Slavic) founder effect. Results of the MLPA analysis indicate that no large exonic deletions are involved in PCD pathogenesis.

Identification and Analysis of Axonemal Dynein Light Chain 1 in Primary Ciliary Dyskinesia Patients

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic infections of the upper and lower airways, randomization of left/right body asymmetry, and reduced fertility. The phenotype results from dysfunction of motile cilia of the respiratory epithelium, at the embryonic node and of sperm flagella. Ultrastructural defects often involve outer dynein arms (ODAs), that are composed of several light (LCs), intermediate, and heavy (HCs) dynein chains. We recently showed that recessive mutations of DNAH5, the human ortholog of the biflagellate Chlamydomonas ODA gamma-HC, cause PCD. In Chlamydomonas, motor protein activity of the gamma-ODA-HC is regulated by binding of the axonemal LC1. We report the identification of the human (DNAL1) and murine (Dnal1) orthologs of the Chlamydomonas LC1-gene. Northern blot and in situ hybridization analyses revealed specific expression in testis, embryonic node, respiratory epithelium, and ependyma, resembling the DNAH5 expression pattern. In silico protein analysis showed complete conservation of the LC1/gamma-HC binding motif in DNAL1. Protein interaction studies demonstrated binding of DNAL1 and DNAH5. Based on these findings, we considered DNAL1 a candidate for PCD and sequenced all exons of DNAL1 in 86 patients. Mutational analysis was negative, excluding a major role of DNAL1 in the pathogenesis of PCD.