ABSTRACT
Functional and ultrastructural abnormalities of respiratory cilia have been described in patients with a congenital respiratory disease known as primary ciliary dyskinesia (PCD) (1,2). PCD, previously described as immotile cilia syndrome, represents a heterogeneous group of genetic disorders affecting 1 in 16,000 indi-
* This work was previously published by the University of Chicago Press in American Journal of Human Genetics 65(6): 1508-1519, 1999. 119
viduals; the PCD phenotype is characterized by impaired mucociliary clearance resulting from a lack of ciliary movements believed to be responsible for chronic lung, sinus, and middle ear diseases. Approximately 50% of the patients with PCD display a situs inversus, thereby defining the Kartagener’s syndrome. Most male patients are infertile because of nonmotile spermatozoa in relation to functional and ultrastructural abnormalities of sperm flagella (3). Studies to date, however, have failed to decipher the molecular basis of this disease. PCD is usually transmitted as an autosomal recessive trait. The various functional and ultrastructural abnormalities of respiratory cilia documented in PCD patients suggest that a genetic heterogeneity underlies this condition (4).The main ciliary defect found in PCD is an absence of dynein arms affecting almost all cilia (3). Dyneins consist of a large family of proteins involved in many types of microtubule-dependent cell motility in both lower and higher eukaryotes. The axonemal dyneins are found in the dynein arms of the ciliary and flagellar axonemes; they are essential for ciliary and flagellar beating.Among the several immotile strains of Chlamydomonas reinhardtii, the mutants carrying a defect in IC78 gene, which encodes a dynein intermediate chain, have been the subject of detailed functional, ultrastructural, and molecular studies (5). Of particular interest, the flagellar ultrastructural phenotype of these mutants is similar to the axonemal ultrastructural abnormality observed in several PCD patients (i.e., absence of outer dynein arms); we have therefore isolated a human gene related to IC78, the DNAI1 gene (6), to test its involvement in PCD. CLINICAL AND ULTRASTRUCTURAL PHENOTYPES OF THE PCD PATIENTS
A 9-year-old Caucasian boy (patient II-1) bom to unrelated parents (family 1) presented in early childhood with chronic respiratory symptoms characterized by chronic sinusitis, serous otitis, and recurrent episodes of bronchitis associated with severe segmental atelectasis, having led to partial lobectomy. Chest radiograph showed normal cardiac and visceral situs. Neither his parents nor other relatives have a history of respiratory disease. At the time of a bronchoscopy, samples of trachea mucosa were obtained and processed for ciliary studies, as described (7). No ciliary beating was observed, and transmission electron microscopy showed the absence of outer dynein arms in all cilia, asserting the diagnosis of PCD (Fig. 1A). Five other unrelated consanguineous PCD families were also investigated (families 2 to 6). In two independent families (families 2 and 3), the ultrastructural phenotype was identical to that documented in patient II-1 from family 1; however, in one case (family 3), the PCD patients also displayed a situs inversus. In the three remaining families (families 4, 5, and 6), both outer and inner dynein arms were absent in the patients’ cilia; in one case (family 6), the disease phenotype was associated with a situs inversus. In all these individu-
als, DNA samples were isolated from peripheral blood samples, according to standard techniques. DNAI1 MUTATIONS LEAD TO A PCD PHENOTYPE
All the coding regions and intron-exon boundaries of the DNA sample from the patient II-1 from family 1 were amplified with DNAI1-specific primers and run on single-strand conformation polymorphism (SSCP) gels or sequenced directly. Two SSCP variants were identified in his genomic DNA; these variants were located in two PCR fragments spanning exon 5 (Fig. 2A) and 1 (Fig. 3A), respectively. To characterize these molecular variations, we cloned the corresponding PCR products. A 4-bp insertion, located at codon 95, was present in 10 of the 20 clones spanning exon 5 (Fig. 2B). A similar experimental approach led to the identification of a 1-bp insertion in the splice-donor site following exon 1 (Fig. 3B). The patient therefore carries two different DNAI1 mutations, thereby demonstrating compound heterozygosity in keeping with the absence of consanguinity documented in this family.The maternal 4-bp insertion results in a frameshift leading to premature stop codon 24 amino acids downstream. To determine the consequences of the paternally inherited splice-donor-site mutation on the processing of DNAI1 transcripts, total RNA obtained from nasal epithelial cells of patient II-1 was reverse-transcribed. The resulting products were used as templates in a PCR assay performed with DNAI1-specific primers (Fig. 3C). Two molecular species were generated: one of expected size (290 bp), and a 422-bp fragment, which was observed only in the patient’s sample and not in the control. Sequencing of these PCR
products (290 bp and 422 bp) revealed that the larger fragment contained a 132-bp insertion corresponding to the 5' intronic sequence following exon 1, indicating the use of a cryptic splice-donor site in this intron at position 133 (Fig. 3D). Indeed, sequence analysis of the intronic region spanning this site revealed the existence of a perfect splice-donor-site consensus sequence at position 133 of intron 1. If translated, this abnormal DNAI1 transcript would result in a premature stop codon at position 73. To further test whether these two mutations are responsible for the PCD phenotype, 50 unrelated control individuals were screened for these DNA11 variations. None of their 100 chromosomes contained such mutations. LOCUS HETEROGENEITY IN PCD
In the course of this study, we identified two intragenic nucleotide polymorphisms: one is located at nucleotide 42 (G > C) of intron 11; the other one is a G-to-A transition at nucleotide 1003 resulting in the V3351 substitution. These two intragenic polymorphisms were used to test the involvement of DNAI1 in the PCD phenotype identified in the five remaining families (families 2-6) in which the patients were bom to consanguineous unions. The genotype at these two loci was characterized by genomic DNA sequencing. In family 2, the two affected children, who displayed an absence of outer dynein arms in all cilia, and the healthy older sister share the same DNAI1 genotype, thereby demonstrating an exclusion of linkage between the DNAI1 gene and the PCD phenotype. In family 5, the two affected children who displayed a PCD phenotype characterized by an absence of both outer and inner dynein arms, carried different DNAI1 genotypes at nucleotide 42 of intron 11. In the three remaining consanguineous families (families 3, 4, and 6), the patients were found to be heterozygous at one or at the two intragenic loci (data not shown). CONCLUSION
We postulated that the human DNAI1 gene, which is homologous to the Chlamy-domonas IC78 gene, was an excellent candidate sequence to investigate in patients with a PCD phenotype characterized by an absence of outer dynein arms. Indeed, Chlamydomonas IC78 protein belongs to outer dynein arms (8), and a lack of outer dynein arms has clearly been documented in the axonemes of Chlamydomonas strains in which the IC78 gene is either deleted or disrupted by a large insertion (5). Two different trans-allelic germline DNAI1 mutations leading to frameshifts were indeed identified in one patient presenting with a PCD phenotype associated with an absence of outer dynein arms. The 4-bp insertion identified on the maternal DNAI1 allele, which probably arose by slippage replication, is predicted to produce a frameshift introducing a premature stop codon located
24 amino acids downstream. The paternal mutation is a splice defect; if translated, the abnormal DNAI1 transcript, which retains the first 132 nucleotides of intron 1, would result in a premature TGA stop codon at position 73. Both the maternal mutation and the paternal mutation should, therefore, generate severally truncated polypeptides lacking 85% and 95%, respectively, of the DNAI1 protein. Taken together, these data highly suggest that such truncated proteins, even if synthesized, could not play their key role in the outer dynein arm assembly. We therefore conclude that these mutations, which were absent from 100 control chromosomes, underlie the ultrastructural phenotype observed in this PCD patient. The documented immotile cilia are also consistent with the absence of outer dynein arms; this may be responsible for mucociliary impairment, which led to the severe chronic respiratory symptoms observed in this patient.We have also identified two nucleotide polymorphisms in DNAI1 that allowed us to demonstrate an exclusion of linkage between the DNAI1 gene and the PCD phenotype in five other families. These linkage data, which provide the first clear-cut demonstration of a locus heterogeneity in this condition, also reveal a higher complexity level of heterogeneity than first expected, since patients with identical ultrastructural defects may have mutations in other still unidentified genes; it is tempting to speculate that these latter genes may encode functional partners of DNAI1 involved in dynein arm assembly. Given the documented clinical, ultrastructural and genetic heterogeneity in PCD, it is, therefore, not surprising that genetic analyses performed in different sets of families did not allow to establish a reproducible genetic linkage between the disease phenotype and a given chromosomal region (9-11). However, in theory this kind of approach could be fruitful if applied to the study of separate families, each being considered individually (12). Nevertheless, such genetic linkage studies have been hampered by the small size of affected families in which male patients are usually infertile. This is the reason why, as shown in the present study, approaches based on the investigation of candidate genes represent powerful alternatives. These candidate genes include the dynein gene family. This study illustrates the use of one particular Chlamydomonas flagellar mutant as an excellent model for axonemal abnormalities observed in PCD; as several other light (13), intermediate (14,15), and heavy chains (16-20) of dynein have been implicated in other Chlamydomonas flagellar mutants, we consider the corresponding genes as good candidates for other subsets of PCD and related developmental diseases. ACKNOWLEDGMENTS
The authors wish to thank M. C. Millepied and M. Couprie (Ecole Superieure d’lngenieurs en Electrotechnique et Electronique (ESIEE)) for their help in determining the ciliary ultrastructural phenotype. This work was supported by grants from the Chancellerie des Universites (legs Poix), the Assistance Publique/
Hopitaux de Paris (CRC96125), and the Universite Paris XII (BQR). GP is therecipient of a fellowship from the Ministere de V Education Nationale, de la Recherche et de la Technologie. REFERENCES
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