Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome)

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).     

The human dynein intermediate chain 2 gene (DNAI2): cloning, mapping, expression pattern, and evaluation as a candidate for primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic sinusitis and bronchiectasis, and usually associated with hypofertility. Half of the patients present a situs inversus, defining the Kartagener's syndrome. This phenotype results from axonemal abnormalities of respiratory cilia and sperm flagella, i.e., mainly an absence of dynein arms. Recently, a candidate-gene approach, based on documented abnormalities of immotile strains of Chlamydomonas reinhardtii, allowed us to identify the first gene involved in PCD. Following the same strategy, we have characterized DNAI2, a human gene related to Chlamzydomonas IC69, and evaluated its possible involvement in a PCD population characterized by an absence of outer dynein arms. DNAI2, which is composed of 14 exons located at 17q25, is highly expressed in trachea and testis. No mutation was found in the DNAI2 coding sequence of the twelve patients investigated. However, ten intragenic polymorphic sites and an EcoRI RFLP have been identified, allowing the exclusion of DNAI2 in three consanguineous families.     

Loss-of-function mutations in a human gene related to Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a group of heterogeneous disorders of unknown origin, usually inherited as an autosomal recessive trait. Its phenotype is characterized by axonemal abnormalities of respiratory cilia and sperm tails leading to bronchiectasis and sinusitis, which are sometimes associated with situs inversus (Kartagener syndrome) and male sterility. The main ciliary defect in PCD is an absence of dynein arms. We have isolated the first gene involved in PCD, using a candidate-gene approach developed on the basis of documented abnormalities of immotile strains of Chlamydomonas reinhardtii, which carry axonemal ultrastructural defects reminiscent of PCD. Taking advantage of the evolutionary conservation of genes encoding axonemal proteins, we have isolated a human sequence (DNAI1) related to IC78, a C. reinhardtii gene encoding a dynein intermediate chain in which mutations are associated with the absence of outer dynein arms. DNAI1 is highly expressed in trachea and testis and is composed of 20 exons located at 9p13-p21. Two loss-of-function mutations of DNAI1 have been identified in a patient with PCD characterized by immotile respiratory cilia lacking outer dynein arms. In addition, we excluded linkage between this gene and similar PCD phenotypes in five other affected families, providing a clear demonstration of locus heterogeneity. These data reveal the critical role of DNAI1 in the development of human axonemal structures and open up new means for identification of additional genes involved in related developmental defects.     

Identification of the human ortholog of the t-complex-encoded protein TCTE3 and evaluation as a candidate gene for primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease that is caused by impaired ciliary and flagellar functions. About 50% of PCD patients show situs inversus, denoted as Kartagener syndrome. In most cases, axonemal defects in cilia and sperm tails can be demonstrated by electron microscopy, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. In order to identify novel PCD genes we have isolated the human ortholog of the murine TCTE3 gene. The human TCTE3 gene encodes a dynein light chain and shares high similarity to dynein light chains of other species. The TCTE3 gene is expressed in tissues containing cilia or flagella, it is composed of four exons and located on chromosome 6q25-->q27. To elucidate the role of TCTE3 as a candidate gene for PCD a mutational analysis of thirty-six PCD patients was performed. We detected five polymorphisms in the coding sequence and in the 5' UTR of the TCTE3 gene. In one patient a heterozygous nucleotide exchange was identified resulting in an arginine to isoleucine substitution at the amino acid level. However, this exchange was also detected in one control DNA. Our results indicate that mutations in the TCTE3 gene are not a main cause of primary ciliary dyskinesia.     

Mutations in SPAG1 Cause Primary Ciliary Dyskinesia Associated with Defective Outer and Inner Dynein Arms

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ∼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders.     

Mutations in Radial Spoke Head Protein Genes RSPH9 and RSPH4A Cause Primary Ciliary Dyskinesia with Central-Microtubular-Pair Abnormalities

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, “9+2”-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.     

Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.     

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.     

Handedness and situs inversus in primary ciliary dyskinesia

...The limbs on the right side are stronger. [The] cause may be ... [that] ... motion, and abilities of moving, are somewhat holpen from the liver, which lieth on the right side. (Sir Francis Bacon, Sylva sylvarum (1627).)Fifty per cent of people with primary ciliary dyskinesia (PCD) (also known as immotile cilia syndrome or Siewert-Kartagener syndrome) have situs inversus, which is thought to result from absent nodal ciliary rotation and failure of normal symmetry breaking. In a study of 88 people with PCD, only 15.2% of 46 individuals with situs inversus, and 14.3% of 42 individuals with situs solitus, were left handed. Because cerebral lateralization is therefore still present, the nodal cilia cannot be the primary mechanism responsible for symmetry breaking in the vertebrate body. Intriguingly, one behavioural lateralization, wearing a wrist-watch on the right wrist, did correlate with situs inversus.     

Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.     

Kartagener's syndrome and the syndrome of immotile cilia

Summary Kartagener's syndrome (KS) is a hereditary disease with typical symptoms of situs inversus, bronchiectasis, and chronic infections of the nasal mucosa. Autosomal recessive inheritance cannot be doubted on account of repeated observations of affected sibs and parental cansanguinity. The bronchopulmonary symptoms in sibs, however, cannot be explained by this mode of inheritance.Recent clinical findings and electron microscope investigations suggest that KS is a special form of manifestation within the immotile cilia syndrome. This disease combines the typical bronchial and nasal symptoms of KS with sterility in the male due to immotile sperm tails and, as a facultative symptom, situs inversus. Thus, sibs with bronchiectasis but without situs inversus are also classified under this syndrome. The symptoms mentioned are caused by an abnormal morphology of bronchial cilia and sperm tails, which can be demonstrated by electron microscopy. The dynein arms normally attached to the nine microtubular doublets and providing a normal ciliary movement are lacking.It is assumed that during early embryonic life ciliary beats in the growing embryo determine the type of laterality. When ciliary movements are absent laterality may develop fortuitously, thus effecting a situs inversus in about half the affected cases. The numerical evaluation of pedigrees from the literature supports this assumption.     

Primary ciliary dyskinesia in mice lacking the novel ciliary protein Pcdp1

Primary ciliary dyskinesia (PCD) results from ciliary dysfunction and is commonly characterized by sinusitis, male infertility, hydrocephalus, and situs inversus. Mice homozygous for the nm1054 mutation develop phenotypes associated with PCD. On certain genetic backgrounds, homozygous mutants die perinatally from severe hydrocephalus, while mice on other backgrounds have an accumulation of mucus in the sinus cavity and male infertility. Mutant sperm lack mature flagella, while respiratory epithelial cilia are present but beat at a slower frequency than wild-type cilia. Transgenic rescue demonstrates that the PCD in nm1054 mutants results from the loss of a single gene encoding the novel primary ciliary dyskinesia protein 1 (Pcdp1). The Pcdp1 gene is expressed in spermatogenic cells and motile ciliated epithelial cells. Immunohistochemistry shows that Pcdp1 protein localizes to sperm flagella and the cilia of respiratory epithelial cells and brain ependymal cells in both mice and humans. This study demonstrates that Pcdp1 plays an important role in ciliary and flagellar biogenesis and motility, making the nm1054 mutant a useful model for studying the molecular genetics and pathogenesis of PCD.     

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.     

Mapping of epidermolysis bullosa simplex mutation to chromosome 12.

Epidermolysis bullosa simplex (EBS) is a dominantly inherited genodermatosis characterized by intraepidermal blister formation. Recent reports have suggested that EBS mutations may relate to keratin abnormalities. In this study, we conducted RFLP analyses to test the hypothesis that EBS is linked to one of the keratin gene clusters on chromosome 12 or chromosome 17. Although these keratin gene loci are not defined by RFLPs, several mapped RFLPs in the same chromosomal regions could be tested for linkage. A large EBS family with 14 affected and 12 unaffected individuals in three generations was analyzed for RFLP inheritance. Within this family there was no evidence for linkage of the EBS mutation to markers on chromosome 17q. However, there was evidence for close linkage to D12S17 located on chromosome 12q, with a maximum LOD score of 5.55 at theta = 0. Mapping of this mutation to chromosome 12 defines an EBS locus distinct from both EBS1 (Ogna) and EBS2 (Koebner), which are on chromosomes 8 and 1, respectively. Further mapping will determine whether this EBS locus on chromosome 12 resides within the keratin gene cluster at 12q11-q13.     

Genetic linkage studies of chromosome 17 RFLPs in von Recklinghausen neurofibromatosis (NF1)

Recent localization of the gene for von Recklinghausen neurofibromatosis (NF1) to chromosome 17 has led to studies to identify additional tightly linked probes that can be used in defining the primary genetic defect in NF1. We have examined and obtained blood for DNA linkage studies on over 250 individuals from 10 multigeneration neurofibromatosis families. We have analyzed 130 members in 7 families with the available chromosome 17 NF1 linked probes, pE51, D17S71, and D17Z1, as well as two probes generated from our own chromosome 17/19 enriched library (LDR92, LDR152A). Tight linkage was found between NF1 and the centromeric probe D17Z1 (theta = 0.04) and between NF1 and D17S71 (theta = 0.08). A definite recombinant was seen for the D17Z1 marker, which previously had not exhibited crossingover. Chromosome 17 DNA markers pE51, LDR92, and LDR152A gave slightly positive scores, which were not statistically significant.     

Exclusion of the phosphatidylinositol-specific phospholipase C β3 (PLC β3) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human phosphatidylinositol-specific phospholipase C β3 (PLC β3) gene, which is located within this region, was considered to be a good candidate for the MEN 1 gene. In this study, the structure and expression of the PLC β3 gene in MEN 1 patients were investigated in more detail, to determine its potential role in MEN 1 tumorigenesis. Southern blot analysis, using blood and tumor DNA from affected persons from seven different MEN 1 families, did not reveal structural abnormalities in the PLC β3 gene. To detect possible point mutations, or other small structural aberrations, direct sequencing of PLC β3 cDNAs from two affected persons from two different MEN 1 families was performed, but no MEN 1-specific abnormalities were revealed. Several common nucleotide sequence polymorphisms were detected in these cDNAs, proving that both alleles of the PLC β3 gene were expressed and analyzed. In conclusion, these results exclude the PLC β3 gene as a candidate gene for MEN 1. Received: 20 March 1996