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Department of Respiratory Diseases, Research Institute, International Medical Center of Japan
Fourth Department of Internal Medicine, Nippon Medical School
Unit of Human Biology and Genetics, Department of Biological Sciences, Graduate School of Science
Department of Human Genetics, Graduate School of Medicine, University of Tokyo
Department of Respiratory Medicine, Respiratory Center, Toranomon Hospital
Department of Pulmonary Medicine, Toho University School of Medicine, Tokyo
Departments of Pathology and Respiratory Medicine, Tenri Hospital, Nara, Japan
ABSTRACT
Diffuse panbronchiolitis (DPB) is a chronic inflammatory airway disease predominantly affecting Asian populations. DPB is considered to be a complex genetic disease. Considering the mucous hypersecretion of the disease, we hypothesized that the transcriptional activity of mucin genes may be altered in DPB. We analyzed nucleotide sequences of regulatory region of six mucin genes—MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7—and detected their promoter polymorphisms. Among them, the insertion/deletion polymorphism identified in the MUC5B gene was significantly associated with the disease (p = 0.0001). Transcriptional activity observed in the three major promoter haplotypes corresponded to the strength of the disease association in which these haplotypes are involved. Immunohistochemistry of the lung tissues of DPB revealed that MUC5B was abundantly expressed not only in bronchial glands but also in increased numbers of goblet cells on the bronchial surface, where MUC5AC is predominant and MUC5B expression is generally scarce in the normal lung. Marked mucous hypersecretion observed in DPB may be partly explained by increased and aberrant expression of MUC5B. The possible involvement of MUC5B gene in DPB was demonstrated. A further role of the MUC5B polymorphism in its pathogenesis should be studied in the future.
Key Words: case-control study disease susceptibility gene expression immunohistochemistry polymorphism
Diffuse panbronchiolitis (DPB) is a disease that was established as a new clinicopathologic entity distinct from chronic obstructive pulmonary disease (COPD) (1). Patients usually suffer from chronic cough and a large amount of sputum and often have a long history of chronic sinusitis. Chronic bacterial infections, later superinfected with Pseudomonas aeruginosa, are often noted during the clinical course of the disease. Infiltrations of lymphocytes, plasma cells, and foamy histiocytes around respiratory bronchioles are delineated as centrilobular micronodules on high-resolution computed tomographic images (2).
Although the etiology of DPB has not yet been clarified, genetic predisposition to the disease has been suspected, because most cases of DPB have been reported from east Asia, and some cases that have been reported outside Asia have been notably among Asian emigrants (3eC6). In Japanese patients, a strong association with HLA-B54, unique to Asians, was reproducibly demonstrated (7, 8). Korean patients with DPB showed a positive association with HLA-A11 (9). Although these findings suggest that one of the susceptibility genes could be located between HLA-A and HLA-B loci, it has not been thus far identified (10).
In addition to the possible HLA-related susceptibility gene, other candidate genes might modify the disease process. Tumor necrosis factor (11), transporter associated with antigen processing (TAP) (12), NADPH/NADH oxidase (13), interleukin 8 (14), and cystic fibrosis transmembrane conductance regulator (CFTR) genes (15) have been studied in the past. Although several variations were identified within these genes, associations with the disease were not corroborated.
Mucous hypersecretion is often observed in various chronic respiratory disorders, such as chronic bronchitis, bronchial asthma, and bronchiectasis. Especially in DPB, patients always suffer from a large amount of sputum. Although more than 95% of the airway secretion consists of water, the main glycoprotein contained in the airway secretion is mucin. We thus hypothesized that the transcriptional activity of mucin genes may be altered in DPB. We examined this possibility by analyzing polymorphisms of the promoter region of six mucin genes—MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7—expressed in the human respiratory tract (16) and attempted to demonstrate its functional significance. Some of the results of these studies have been previously reported in the form of abstracts (17eC18).
METHODS
Subjects
Blood samples were obtained from 92 unrelated Japanese patients with DPB, which was diagnosed according to the criteria proposed in 1995 by the Working Group of the Ministry of Health and Welfare of Japan (2). As a control population, 128 individuals were anonymously selected at random from the general Japanese population. As a second control, 147 healthy Japanese individuals were further analyzed. Genomic DNA was extracted by a method described elsewhere (19). Blood samples from 40 healthy Caucasian individuals were also tested in this study. Furthermore, 10 chimpanzee DNA samples were analyzed to evaluate a possible history of evolution on polymorphisms.
Informed consent was obtained from all the subjects, and this study was reviewed and approved by the Joint Ethical Committee of the International Medical Center of Japan. The collection of chimpanzee blood samples was approved by the Institutional Animal Welfare Committee, Primate Research Institute, Kyoto University.
Direct Sequencing for Identification of Polymorphisms and Determination of Their Genotype Frequencies
Promoter regions of MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7 genes were screened for discovery of variations in 16 unrelated healthy Japanese. On the basis of the previous reports that had examined the promoter activity of these mucin genes, the regions to be analyzed were then selected (20eC27). Polymerase chain reaction primer sets were designed to cover these regions, and polymerase chain reaction products were directly sequenced using an ABI 3100 automated sequencer (Applied Biosystems, Foster City, CA).
Single-Strand Conformation PolymorphismeCbased Direct Haplotype Determination Method
Among eleven polymorphisms identified in the promoter region of the MUC5B gene, six consecutive polymorphisms located near the transcription start site have been known to be in strong linkage disequilibrium (28). To assess the difference of haplotype frequencies between cases and control subjects accurately, we determined haplotypes of the region directly using the single-strand conformation polymorphismeCbased direct haplotype determination (SSDHD) method reported elsewhere (28).
Single-Strand Conformation Polymorphism Analysis for Genotyping of Insertion/Deletion Polymorphism in the Promoter Region of the MUC5B Gene
In addition to the direct sequencing, the dinucleotide insertion/deletion polymorphism in the promoter region of the MUC5B gene was also genotyped applying the conventional single-strand conformation polymorphism (SSCP) method.
Construction of Promoter Reporter and Transient Transfection
A DNA region encompassing three major haplotypes in the 5'-flanking region of the MUC5B gene was amplified by polymerase chain reaction and then cloned into a pGL3 basic vector (Promega, Madison, WI; Figure 1). The plasmids were transfected into NCI-H292 cells (American Type Culture Collection number CRL-1848), and the cells were harvested after 24 hours. Luciferase reporter gene activity was determined using the Dual-Luciferase Reporter Assay System (Promega).
Immunohistochemistry Using Anti-MUC5AC and Anti-MUC5B Antibodies
We used autopsy samples of the lung tissues of patients with DPB and those without any obvious lung diseases. COPD samples were also analyzed. Patients' clinical information is shown in Table 1.
For immunostaining, one of the following primary antibodies was applied: anti-MUC5AC mouse monoclonal antibody (1:1000 dilution; Clone 45M1; NeoMarkers, Fremont, CA) or anti-MUC5B rabbit polyclonal antibody (1:400 dilution; raised against a recombinant protein corresponding to amino acids 1201eC1500 of MUC5B; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After overnight incubation at 4°C, secondary antibodies labeled with peroxidase were applied. Color development was performed using 3-amino-9-ethyl carbazole liquid substrate chromogen (Dako, Glostrup, Denmark).
Phylogenetic Tree Analysis
The haplotype tree was constructed from the nucleotide sequences of MUC5B gene (nt 3195eC4203 in AF107890) of five haplotypes most frequently observed in Japanese and Caucasians, respectively. The most frequent haplotype observed in the corresponding sequences in chimpanzees was incorporated into this analysis. The construction of an unrooted tree by the neighbor-joining method was performed using the PHYLIP 3.6 neighbor program (29, 30).
Statistical and Computational Analyses
Conformity of the genotype distribution of the polymorphisms to the Hardy-Weinberg equilibrium was examined in the studied population using the program Arlequin (version 2.000) (31). Disease associations with each polymorphism were assessed by 2 test. A p value of less than 0.05 was considered to be significant. Frequencies of haplotypes containing multiple polymorphic sites were estimated by the PHASE program (version 2.1) (32, 33). A case-control permutation test using estimated haplotypes between patients with DPB and healthy Japanese was also performed by PHASE. An analysis of variance was performed on the difference of luciferase activity using JMP (version 5; SAS Institute, Inc., Cary, NC). Putative transcription factor binding sites were extracted using MatInspector (Genomatix, Munich, Germany) (34).
RESULTS
Identification of Polymorphisms in the Promoter Region of Six Mucin Genes
We investigated the promoter region of mucin genes MUC1, MUC2, MUC4, MUC5AC, MUC5B, and MUC7, which are expressed in human airways. No polymorphism was found in the promoter region of the MUC1 gene. In the promoter regions of MUC2, MUC4, MUC5AC, and MUC7 genes, two to four polymorphisms were found. In contrast, in the promoter region of the MUC5B gene, we identified 10 single nucleotide polymorphisms (SNPs) and a dinucleotide insertion/deletion polymorphism, which were distributed in almost every 180 bp in the 2-kb promoter region of the MUC5B gene. The polymorphisms identified in each mucin gene are shown in Table 2.
Disease Association with MUC5B Gene Polymorphisms
A total of 22 polymorphisms identified in the promoter region of five mucin genes were genotyped in cases and control subjects. Only three polymorphisms in the promoter region of the MUC5B gene showed significant associations with the disease (Table 3). Of these, a dinucleotide CA insertion/deletion polymorphism showed the strongest association with the disease when the allele frequencies were compared (p = 0.0001), and the individual genotypes were further verified by polymerase chain reactioneCSSCP analysis (Figure 2). We tested this association by using the second controls of 147 healthy individuals. In the second control subjects, the frequency of the D allele was 0.262, comparable to that of the first control group, and it was significantly different from that of the patient population (p < 0.0001). In addition, the frequencies of the polymorphism were not different between males and females (data not shown). Even after the p value had been multiplied by the number of polymorphisms analyzed in this study, this association remained significant. None of the genotype distribution in the control population deviated from expectations based on the Hardy-Weinberg equilibrium (data not shown).
Direct Determination of Haplotypes in the Promoter Region of the MUC5B Gene and Their Disease Association
To compare haplotype frequencies directly between cases and control subjects, we determined haplotypes experimentally using long polymerase chain reaction products encompassing six consecutive polymorphic loci near the transcription start site by the SSDHD method. The frequencies of directly determined haplotypes by this method are listed in Table 4. Thirteen haplotypes were identified among the Japanese population. Of these, three major haplotypes whose allele frequencies were more than 10% were identified in the control population, and these three haplotypes accounted for approximately 80% of the alleles. H1 and H3 contain insertion of CA and H2 contains deletion of CA. H2 was significantly decreased in patients with DPB (p = 0.0002). Although H1 was increased in the patients, the difference did not reach the significant level (p = 0.063).
The difference of estimated haplotype distribution was also compared between cases and control subjects by the permutation test using the PHASE program. This was statistically significant (p = 0.03). Haplotype frequencies estimated by the PHASE method were in good accordance with those obtained by direct haplotype determination (data not shown).
Genotyping and Haplotype Estimation of Caucasians and Construction of a Phylogenetic Tree
We sequenced the same region in 40 Caucasians. Although no dinucleotide insertion/deletion polymorphism was identified among them, positions of five other polymorphic sites were the same as those found in the Japanese. In addition to the five SNPs, a rare SNP (A/G) with a minor allele frequency lower than 2% was identified at position 3974 of AF107890. An estimation of haplotype frequency revealed that H3 was the most frequent haplotype among Caucasians (C1), with an estimated frequency of 76.3%. The haplotype H1, which was most frequent among the Japanese, was estimated to be only at 6.0% in Caucasians (C3), and the second frequent haplotype H2 was not found in the population.
Ten chimpanzee samples were also sequenced. In chimpanzees, six different polymorphic sites were identified. The polymorphic sites found in human samples were not identified. Six haplotypes were estimated in the chimpanzees.
Using five representative haplotypes among Japanese and Caucasian populations and one that was the most frequent haplotype accounting for 40% of chimpanzee alleles, we constructed an unrooted phylogenetic tree (Figure 3). The chimpanzee sequence was used as an out-group. Haplotypes of humans are remotely related to those of chimpanzees, and they seem to have emerged in humans according to the process of evolution from a common ancestor. The haplotype H2, which was found to have the second frequency only among the Japanese, detached itself from the branch of H4 haplotype after having created several branches.
Transcriptional Activity of the Three Major Promoter Haplotypes of the MUC5B Gene
To determine whether the transcriptional activities are affected by the difference of haplotypes, the luciferase assay was performed. The DNA sequences that encompass three major haplotypes identified in the promoter region of the MUC5B gene were cloned into the vectors, and those plasmids were transfected into NCI-H292 cells that express MUC5B. Relative luciferase activity in the steady state was measured.
As shown in Figure 4, the transcriptional activity of the clone containing H2 was the lowest, H3 was intermediate, and H1 was the highest.
Immunohistochemistry
Immunohistochemistry for two major airway-secreted mucins, MUC5AC and MUC5B, was performed on formalin-fixed paraffin-embedded lung tissues from autopsied cases of DPB and others. Each primary antibody is known to recognize the peptide core of the corresponding mucin, and the staining patterns of normal tissues are consistent with those reported by others (26).
In lung tissues from an autopsied case without any obvious airway diseases (Lu-1, as shown in Table 1), immunohistochemistry for MUC5AC showed staining restriction in goblet cells of the bronchial surface epithelium (Figure 5A), and submucosal gland cells were mostly negative in staining. Similar results were obtained from another case without any obvious airway diseases (Lu-2). On the other hand, in a patient with DPB [DPB-1(2)], goblet cells of bronchial epithelium were increased and more than 90% of goblet cells were positive for MUC5AC (Figure 5B). MUC5AC immunoreactivity was not observed in submucosal gland cells. Similar results were also observed on the tissue sections from other samples [DPB-2(1), DPB-3(2), DPB-4(1), and DPB-4(2)]. The remaining DPB samples listed in Table 1 are mainly from peripheral lung tissues; therefore, it was not possible to evaluate large airways in these samples.
Immunohistochemistry for MUC5B in the serial section of the lung tissue without obvious airway disease (Lu-1) showed the immunoreactivity mainly in the submucosal glands and no obvious staining was observed in surface goblet cells (Figure 6A). Exceptionally, the immunoreactivity shown for MUC5B was sparsely observed in cells of the airway surface in some fields (Figure 6B). A similar result was also observed in Lu-2. In contrast, MUC5B was strongly stained in submucosal gland cells and abundantly observed in surface goblet cells of DPB samples [DPB-1(2); Figures 6C and 6D]. Submucosal glands seemed to be hypertrophic compared with those without any obvious lung diseases. Consistently, immunohistochemistry of MUC5B in three other DPB cases [DPB-2(1), DPB-3(2), DPB-4(1), and DPB-4(2)] showed similar results.
Immunoreactivity for MUC5AC and MUC5B in respiratory bronchioles of DPB samples was examined. Both MUC5AC and MUC5B were negative on the surface epithelium of respiratory bronchioles (Figure 7). The accumulation of foamy macrophages and lymphocytes in interstitium around respiratory bronchiole was noted, and some of the airway lumens were filled with MUC5B-positive secretions (Figure 7B).
We also performed immunohistochemical studies in three COPD samples. Localization of MUC5AC immunoreactivity in the lung tissues of COPD-1 and COPD-2 are shown in Figures 8A and 8B, respectively. MUC5AC immunoreactivity was restricted to surface goblet cells, and the number of MUC5AC-positive goblet cells increased more than in normal samples. The localization of MUC5B in serial sections of the same two cases is shown in Figures 8C and 8D. The submucosal gland was enlarged and many mucous cells were positive for MUC5B.
Surface goblet cells were negative for MUC5B in COPD-1 (Figure 8C); however, in COPD-2 (Figure 8D), many goblet cells were positive for MUC5B staining. Thus, MUC5B immunoreactivity in surface goblet cells showed that it was not homogeneous in patients with COPD.
DISCUSSION
In the present study, we initially performed comprehensive genetic analyses of the promoter region in six mucin genes mainly expressed in human airways. Of 22 polymorphisms screened, three polymorphisms of the MUC5B gene showed significant association with DPB. The strongest association was observed in a dinucleotide insertion/deletion polymorphism, which was identified 657 to 658 bp upstream of the transcription start site.
Most of the human mucin genes show length polymorphism, which means polymorphism of various lengths because of variable numbers of tandem repeat in the coding sequence (35). For example, each unit of tandem repeats of the MUC1 gene encodes 20 amino acids, and the variable numbers of tandem repeat polymorphism can be detected as genomic DNA fragments by the digestion with restriction enzyme, with the fragments ranging from 2.8 to 8.0 kb (36, 37). The length variation of mucins is likely to have impact on the properties of the mucus gel and may lead to functional difference. However, the MUC5B gene has been reported to show little variation in variable numbers of tandem repeat and this indicates that the interindividual difference of MUC5B property may be scant compared with other mucins (38). Instead, as shown in this study, relatively large numbers of polymorphisms in the promoter region of the MUC5B gene were identified, which implies that the interindividual difference of MUC5B is mainly determined at expression levels.
Because three polymorphisms in the promoter region of the MUC5B gene showed significant associations with DPB, we performed haplotype analysis using an experiment containing three polymorphisms. The haplotype estimation program also accurately estimated haplotype frequencies and differences between cases and control subjects, because six polymorphisms were in relatively strong linkage disequilibrium. The haplotype H2, which contains deletion of CA, showed a significant negative association with DPB. This deletion allele was not observed in Caucasian samples examined, and the most frequent haplotype H1 showing marginal positive association with the disease in Japanese accounted for less than 10% in Caucasian individuals. Thus, it was revealed that frequencies of major haplotypes were remarkably different between the two ethnic populations. Chimpanzees had quite different polymorphic sites from those of humans and also lacked the deletion allele that was observed in the Japanese population. On the basis of phylogenetic tree analysis, it seems that the major haplotypes observed both in Japanese and Caucasians appeared after the evolution to humans, and that the H2 haplotype, unique to Asians, subsequently developed.
The luciferase assay clearly demonstrated that transcriptional activity was significantly different among three major promoter haplotypes of the MUC5B gene, and the luciferase activity of haplotype H2 was the lowest. H3 was intermediate and H1 was the highest. Thus, transcriptional activity of the three major haplotypes at a steady level was parallel to the strength of association. Considering the results of luciferase analysis, the deletion allele might be working as a resistant factor to the disease by decreasing the transcription activity, and the insertion allele is considered to be more susceptible to mucous hypersecretion than the deletion allele.
Although the expression of the MUC5AC gene has been reported to be increased by various stimuli, the signaling system that could upregulate MUC5B gene transcription has not yet been clarified. The insertion/deletion polymorphism is incorporated into a consensus sequences of binding sites of vitamin D receptor/retinoid X receptor heterodimer site, interferon (IFN)-stimulated response element, and signal transducer and activator of transcription 6 (STAT6) by computer estimation of transcription factor binding sites. Regarding the retinoid X receptor (RXR), it has been reported that retinoid acid receptors (RARs) and RXRs can act synergistically in the induction of the MUC2 gene and MUC5AC gene expression (39), although induction of MUC5B gene expression has not been mentioned. STAT6 has been reported to upregulate MUC2 by the signaling of IL-4 and IL-13 (40). There has been so far no report that IFN- or IFN- has any role in the regulation of transcription of mucin genes.
Considering the role of MUC5B in DPB further, we examined distribution of the expression in the lung by immunohistochemistry. In normal lung tissues, the expression of MUC5AC, one of the two major secretory mucins in human airways, was restricted to goblet cells on the airway surface, and that of MUC5B, the other major mucin, was found mainly in submucosal gland cells and in a few goblet cells. Tissue distribution of MUC5AC and MUC5B expression was consistent with the findings of the previous reports in which the expression of these mucins was detected using in situ hybridization (26) and by immunohistochemical study (41).
In all DPB cases tested, increased numbers of goblet cells and enlarged submucosal glands were observed, presumably causing mucous hypersecretion. MUC5AC was abundantly expressed in surface goblet cells, although it was not expressed in submucosal gland cells, which indicates that tissue distribution of MUC5AC itself was not altered in DPB. The overproduction of MUC5AC core protein in bronchoalveolar lavage fluid among patients with DPB was reported recently (42). On the other hand, MUC5B was expressed not only in submucosal gland cells but also in surface goblet cells. Thus, distribution of MUC5B expression was markedly altered in DPB. Because almost all the goblet cells were positive for the two major mucins, as shown in immunostaining, goblet cells secreting MUC5AC may simultaneously secrete MUC5B in this disease. In addition, MUC5B expression was predominantly observed in the large airway. In the pathogenesis of DPB, the relationship between hypersecretion in the large airway and chronic inflammation in the respiratory bronchioles still remains unknown. In the immunohistochemical study on COPD samples, MUC5B expression was increased both in submucosal gland cells and surface goblet cells. However, the increase was much less prominent compared with that in DPB. The possibility that expression of MUC5B is increased in surface goblet cells in inflammatory lung diseases has been previously reported (26). Our observations support these findings. The mechanisms for aberrant expression of MUC5B gene in the inflammatory state are unknown. Although future studies are necessary to find out whether genetic polymorphisms identified in this study are directly involved in the MUC5B expression at sites of the disease, our present study will provide clues to uncover the role of MUC5B in the disease process, which so far has been almost ignored.
In conclusion, the level of mucus secretion might be modified by single variation or haplotypes in the promoter region of the MUC5B gene. A possibility of MUC5B gene involvement in DPB was demonstrated on the basis of molecular genetics, in vitro functional analysis, and tissue distribution. Further evidence that the MUC5B polymorphism plays a role in the pathogenesis of DPB is awaited.
Acknowledgments
The authors thank Kazuko Tanabe, D.V.M., for her critical reading of the manuscript and Ms. Kyoko Hasegawa and Ms. Tokie Totsu for their technical support.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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