Published ahead of print on November 20, 2003, doi:10.1165/rcmb.2003-0338RC
© 2004 American Thoracic Society DOI: 10.1165/rcmb.2003-0338RC
Investigation of the Possible Role of a Novel Gene, DPCD, in Primary Ciliary DyskinesiaDepartments of Medicine, Pathology, and Pediatrics, and the Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Address correspondence to: Lawrence E. Ostrowski, Ph.D., University of North Carolina at Chapel Hill School of Medicine, Cystic Fibrosis/Pulmonary Research and Treatment Center, CB# 7248, 6123A Thurston-Bowles Bldg., Chapel Hill, NC 27599-7248. E-mail: ostro{at}med.unc.edu
Primary ciliary dyskinesia (PCD) is an autosomal recessive disease caused by mutations that affect the proper function of cilia. Recently, deletion of DNA polymerase  (Poll) in mice produced a phenotype characteristic of PCD (Kobayashi et al., 2002, Mol. Cell. Biol. 22:2769–2776). Because it is unclear how a mutation in a DNA polymerase would result in a specific defect in axonemes, the targeting construct was examined further. Analysis of the genomic region surrounding the Poll gene revealed an uncharacterized gene, named Dpcd, that is predicted to be transcribed from the opposite strand relative to Poll. The deletion of Poll would also remove the first exon of Dpcd. Because it is possible that the PCD phenotype observed is due to the absence of either gene, the expression of these genes during ciliogenesis of human airway epithelial cells was examined. Northern analysis demonstrated that DPCD expression increases during ciliated cell differentiation; the expression of POLL decreases. To examine directly whether DPCD is mutated in cases of human PCD, the complete coding sequence of DPCD was sequenced from 51 unrelated PCD patients. No disease-causing mutations were confirmed; however, one variant could not be excluded. Therefore, DPCD remains a novel candidate gene for PCD.
Abbreviations: deleted in a mouse model of PCD, Dpcd • human bronchial epithelial cells, HBE cells • inner dynein arm, IDA • outer dynein arm, ODA • primary ciliary dyskinesia, PCD • DNA polymerase
Primary ciliary dyskinesia (PCD) is a genotypically heterogeneous disease, usually inherited as an autosomal recessive trait, caused by mutations that impair the function of cilia and flagella (1–3). The defect in ciliary function in the respiratory tract results in impaired or absent mucociliary clearance, which is believed to be responsible for the frequent and recurrent episodes of sinusitis and bronchitis that are typical of this disease. The phenotype of the disease is also variable; in severe cases patients develop end-stage bronchiectasis and require lung transplantation. In addition, the disease affects other organs and processes that require ciliary or flagellar motion, and patients with PCD may exhibit otitis media, situs inverses totalis, and/or infertility.
The respiratory cilium is a complex structure consisting of
Recently, a deletion of DNA polymerase To investigate the possible role of DPCD in human cases of PCD, the expression of DPCD was examined in a panel of human tissues to determine if the gene was expressed in a specific pattern consistent with the PCD phenotype. The expression of DPCD and POLL were also examined during ciliogenesis of human bronchial epithelial (HBE) cells in vitro. Finally, to examine whether mutations in DPCD or POLL could account for some cases of human PCD, we have sequenced the coding regions of both DPCD and POLL in a cohort of well-characterized PCD patients. A preliminary report of this work has been presented (14).
Database Analysis The Dpcd gene was found in the Celera Discovery System mouse genomic database by searching the region surrounding Poll. Subsequent analysis identified the same gene in the National Center for Biotechnology (NCBI) databases (http://www.ncbi.nlm.nih.gov). Ensembl (http://www.ensembl.org) was used to visualize the gene relative to Poll on the chromosomes of mouse and human.
Analysis of DPCD and POLL Expression
Clinical Evaluation and Ciliary Dynein Arm Evaluation of Patients with PCD
Mutation Profiling
Analysis of mouse chromosome 19 in the region of the Poll gene revealed an uncharacterized gene (mCG127478 in the Celera mouse genomic database) predicted to be transcribed from the opposite strand relative to Poll. The predicted first exon of this novel gene, named Dpcd, lies only 75 bases from exon 1 of Poll, in the same region encompassed by the deletion of Poll reported to produce a PCD phenotype (12). The 6.4-kb deletion is predicted to remove the first exon of Dpcd, including the predicted first ATG, and therefore would likely disrupt the function of both Dpcd and Poll (Figure 1). Thus the phenotype observed could be due to the loss of either gene. For comparison, the deletion reported by Bertocci and colleagues (13) is also shown. This deletion, which replaced three exons of pol with a neo gene, did not produce a PCD phenotype, suggesting the disruption of Dpcd is likely responsible for the PCD phenotype.
The human homolog of Dpcd (accession number XM_0583581; designated DPCD) is located in a highly syntenic region of chromosome 10 (Figure 1). The predicted human DPCD protein (NP_056263) is 88% identical and 94% similar to the mouse protein. DPCD is predicted to code for a novel protein of 23 kD with no homology to any conserved protein domains. Because PCD typically affects a specific set of axoneme containing tissues, including the airways and reproductive organs, we examined a panel of human tissues to determine the pattern of DPCD expression. Probing northern blots containing samples from 16 different human tissues showed that the highest level of DPCD expression was in the testis (Figure 2). A single band close to the expected size of 821 bp was observed, indicating the absence of any major splice variants. Weak signals were also observed in RNA from pancreas, skeletal muscle, and heart. Other tissues examined showed no detectable expression, even after prolonged exposure (14 d). The expression of POLL is also highest in testis, with lower levels of expression reported in ovary, skeletal muscle, pancreas, and heart (18–20). Thus the expression pattern of DPCD and POLL follow a similar pattern when analyzing RNA extracted from total tissues. However, the absence of a DPCD signal in lung and other tissues may be due to the low percentage of ciliated cells in these total tissue samples. To determine if DPCD was expressed in airway epithelial cells, RT-PCR was used to amplify a full-length cDNA from cultured HBE cells. The sequence of the cDNA was identical to that in the database, verifying the expression of the predicted transcript (not shown). To evaluate more specifically whether DPCD and/or POLL are expressed in ciliated airway epithelial cells and whether their expression correlated with ciliogenesis, HBE cells were grown at an air–liquid interface (15, 16). RNA was isolated from parallel cultures at different stages of differentiation and analyzed by Northern blotting. At early time points in these cultures, the HBE cells are undifferentiated and resemble basal cells. With time, the cells differentiate to form a heavily ciliated epithelium similar to that observed in vivo. The expression of ciliated cell–specific genes has previously been shown to correlate with the development of ciliated cells in this model system (e.g., Figure 5 in Ref. 21). The results demonstrate that DPCD is expressed in airway cultures and that it becomes more abundant during ciliated cell differentiation, consistent with a potential role in cilia structure or function (Figure 3, top panel). Reprobing of the blot with a POLL-specific probe showed a low level of expression early in culture, which appeared to decrease as differentiation occurred (Figure 3, middle panel).
To examine whether mutations in DPCD could account for some cases of human PCD, the coding region was sequenced from a group of 68 patients with well-characterized PCD. Initially, we sequenced the coding region of DPCD from 51 unrelated patients with PCD. During the course of sequence analysis, six sequence variants were detected (Table 1).
To determine if any of these variants could be pathogenic, a population study was performed in individuals without PCD (Table 2). Four of these variants were found to be present in individuals without PCD, with varying frequencies. Five non-PCD individuals were homozygous for 467T>C, demonstrating that this variant is clearly not pathogenic. IVS2+59A>G and 399C>T (N133N) variants were not detected in a homozygous state in the control population; however, given their high minor allele frequencies (30% and 22%, respectively), they are most likely polymorphisms. Also, the 399C>T and IVS2+59A>G variants were found to be in a homozygous state in the unaffected sibling of PCD1 (Figure E1 in the online supplement). The unaffected father of PCD151 was also homozygous for the 399C>T variant. IVS2–22_40 del had a minor allele frequency of only 1% in the normal population, but this variant was not detected in an affected sibling of PCD163 (Figure E1 in the online supplement). Therefore, this variant is most likely a rare polymorphism. IVS5–48_50 del was not found in the 106 alleles analyzed from the disease-free individuals. However, it also is probably a rare polymorphism because it was detected in one allele from an affected sibling of PCD354, who was wild-type at this locus (Figure E1 in the online supplement). The 168T>G (S56R) variant was not detected in 100 alleles analyzed from the control population. The role of this variant is less certain because it was detected in a heterozygous state in only one affected individual (PCD222), who was wild-type for all the other coding nucleotides and intron–exon junctions in this gene. It is possible that a mutation in the noncoding region of the other allele of DPCD may be responsible for PCD in this individual. Further studies will be required to test this hypothesis.
The discovery of intragenic polymorphisms was useful for carrying out exclusion mapping analysis to exclude the DPCD gene from linkage to PCD, assuming an autosomal recessive mode of inheritance. For PCD1, affected and unaffected siblings shared the same genotype at the polymorphic loci in intron 2 (IVS2+59A>G) and in exon 4 (399C>T), and both the parents were heterozygous. Thus DPCD was excluded from linkage to PCD in this family. PCD373 was borne of consanguineous union and was heterozygous for IVS2+59A>G; therefore, linkage was also excluded in this family (Figure E1 in the online supplement). For PCD Patients 10, 16, 34, 108, 163, and 354, the affected and unaffected siblings were discordant for either one or more polymorphic loci, hence these families were also excluded. Given the proximity of the POLL gene to DPCD gene, all the above-mentioned families are probably excluded for POLL gene as well. Because the ultrastructural analysis of the Poll knockout mouse revealed an absence of IDA (12), we sequenced the coding region of the POLL gene in a patient from all 13 PCD families with solely an IDA defect, and in two patients with PCD with normal dynein arms. During the course of sequence analysis, eight sequence variants were detected (Table E1 in the online supplement); all of these have been identified as intragenic polymorphisms (Table E2 in the online supplement).
Although a small number of mutations have recently been identified as causes of PCD, in the majority of cases the underlying mutation is still unknown. Recently, a deletion of DNA polymerase was reported to produce a PCD phenotype in a mouse model (12). The COOH terminal region of DNA polymerase (pol ) shows 33% sequence identity with DNA polymerase ß, and recombinant pol displays DNA polymerase activity (19, 20). In addition, a pol -GFP fusion protein has been shown to localize predominantly to the nucleus (20). Because it is difficult to reconcile the function of a nuclear DNA polymerase with a defect in axonemal structure (missing IDA), we examined the deletion construct in more detail and determined that another gene was also likely disrupted in the mouse model. Other reports have also documented the disruption of more than one gene in mouse models (22, 23). Therefore, the results from any genetically modified animal should be interpreted with caution, especially if the phenotype observed does not agree with the known function of the targeted gene. Interestingly, an independent deletion of Poll targeting the catalytic domain produced mice with a normal phenotype (13). This deletion would not be predicted to disrupt the coding sequence of the novel gene. Thus the PCD phenotype is most likely due to disruption of the novel gene. The novel gene, which we have named Dpcd, is predicted to code for a protein of 23 kD of unknown function. Searches of databases as well as protein analysis programs have not revealed any significant homology to known proteins or any conserved functional domains to date. The predicted protein contains several potential phosphorylation sites and potential amidation and glycosylation sites, but further studies of the location and function of this novel protein are clearly required. Because PCD is caused by an inherited defect in the structure or function of cilia, a candidate gene would most likely be expressed in tissues that contain cilia or flagella. DPCD and POLL are expressed at high relative levels in the testis, which is consistent with a possible role in the proper function of cilia and flagella. Both DPCD and POLL are expressed at low levels in a wide variety of other tissues, based on this and previous studies and EST data (18–20). However, in cultures of normal HBE cells undergoing ciliogenesis, DPCD increases in a pattern consistent with other cilia-specific genes (21, 24), whereas the expression of POLL remains constant or decreases. These data suggest that DPCD plays a role in the formation or function of ciliated cells. Because the above data suggest that DPCD may be responsible for some cases of PCD, we examined a group of PCD patients for mutations in DPCD. Thirteen of these patients had a defect in the IDA, which is the same ciliary abnormality observed in the mouse model, and 20 patients had defects in both the IDA and ODA. Six sequence variants were identified, but none were confirmed as disease causing. Although several PCD patients were homozygous for the IVS2+59A>G and 399C>T variants, based on exclusion analysis, these are most likely polymorphisms and not disease causing. In addition, RT-PCR was performed on three patients (PCD 1, 157, and 158) and yielded a product of normal size with no mutations detected (not shown). Although no causative mutations were positively identified in DPCD, one variant, 168T>G, which results in a serine to arginine change, cannot be eliminated without further studies. It is possible that in this patient a mutation in the other DPCD allele may have occurred outside the region sequenced. For example, a mutation in the promoter region may prevent expression of the wild-type allele, creating a DPCD null phenotype. The fact that no disease causing mutations were identified in DPCD is not unexpected. PCD is known to be a heterogeneous disease, and only small numbers of samples are available for analysis. Further, mutations that cause PCD in mouse models may be different than those responsible for human disease. It is interesting to note that hydrocephalus is a common feature of many animal models of PCD, but is uncommon in the human disease. It is possible that mutations in genes like DPCD may not be compatible with life, and so may not be found in the PCD population. A subset of the patients with PCD, including thirteen with only an IDA defect, were also examined for possible mutations in the POLL gene. No mutations were detected in the coding region or splice junctions. A recently reported study also failed to identify mutations in the POLL gene in patients with PCD (25). This is perhaps not surprising, as it appears unlikely that the PCD phenotype observed by Kobayashi and coworkers (12) was caused by the disruption of the Poll gene. In conclusion, our results strongly suggest that the deletion of Dpcd may be responsible for the PCD like phenotype in the mouse model previously reported. This conclusion is supported by the expression pattern of Dpcd, the absence of a known function of a DNA polymerase in the structure/function of cilia, and by the lack of a PCD phenotype in mice in which the catalytic region of Poll was specifically deleted. Our results also show that mutations in the coding region and splice junctions of DPCD do not account for a large percentage of PCD cases, although it is possible that isolated cases may still be found. Further studies are needed to identify the role of DPCD in cilia structure and function.
The authors thank Drs. J. Carson, A. Sannuti, M. Hazucha, and S. Minnix for their help in evaluation of the patients with PCD in this study, and Drs. S. Bell, R.U. De Iongh, and L.M. Morgan for providing additional PCD samples. They also thank L. Bauer, B. Brighton, and K. Burns for excellent technical support, L. Brown for help with the figures, and the patients with PCD for their willingness to participate in this study. This work was supported in part by NIH grant HL63103 (L.E.O.), 5-K23-HL04225 (P.G.N.), RR00046–43 (M.R.K.), Cystic Fibrosis Foundation grant R026-CR02 (W.K.O.), and UNC grant RR00046.
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org
* These authors contributed equally to this work, and are considered co-first authors.
1 This sequence record has been removed from the NCBI database (note added in proof). Received in original form September 12, 2003 Received in final form November 14, 2003
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Abstract
Introduction
250 proteins (
