A common MUC5B promoter polymorphism and pulmonary fibrosis

Abstract

Background: The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk.

Methods: Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue.

Results: Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P=1.2×10(-15); allelic association with idiopathic pulmonary fibrosis, P=2.5×10(-37)). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis.

Conclusions: A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dysregulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.).

Figure 1. Summary of Genetic Screen in Familial Interstitial Pneumonia and Idiopathic Pulmonary Fibrosis

In a linkage analysis involving 82 multiplex families with familial interstitial pneumonia, 884 markers were tested across the genome. Panel A shows the location of the linkage peak on chromosome 11 (maximum LOD score, 3.3) and the adjacent area of fine mapping (red box). Panel B shows the results of fine mapping of the chromosome 11 linkage peak. A total of 306 tagging SNPs were typed across the linkage peak in 145 subjects with familial interstitial pneumonia, 152 subjects with idiopathic pulmonary fibrosis, and 233 healthy controls. Chromosomal location (in megabases) is shown on the x axis. The resequencing diagram in Panel C shows the entire genomic region from MUC2 to MUC5B, drawn to scale except where the dotted line appears. The green, red, and blue blocks represent gene exons, and the orange blocks putative promoter regions. The black blocks represent intergenic regions, and the black horizontal line represents introns. SNPs identified by means of resequencing and tested for association in 54 controls who were spouses of subjects, 96 subjects with sporadic idiopathic pulmonary fibrosis, and 69 subjects with familial interstitial pneumonia are displayed in Panel B as dots. SNPs genotyped in an independent group of 83 subjects with familial interstitial pneumonia, 492 subject with sporadic idiopathic pulmonary fibrosis, and 322 controls are shown in Panel D. In Panels B, C, and D, the results of allelic association testing for each SNP are denoted by red dots for familial interstitial pneumonia and by black dots for idiopathic pulmonary fibrosis. (See the Supplementary Appendix for a description of gene models of gel-forming mucins.)

Figure 2. Pairwise Linkage Disequilibrium Plot for Single-Nucleotide Polymorphisms (SNPs) Significantly Associated with Idiopathic Pulmonary Fibrosis or Familial Interstitial Pneumonia in a Genetic Screen of Lung-Expressed Gel-Forming Mucins

The linkage-disequilibrium values displayed were calculated with the use of the r² statistic for the mucin genetic screen performed in 492 subjects with idiopathic pulmonary fibrosis. The bar above the plot indicates the approximate location of these SNPs within the gel-forming mucin region. The highly significant MUC5B promoter SNP (rs35705950) and its pairwise linkage-disequilibrium values are outlined in red. Linkage-disequilibrium patterns were qualitatively similar among controls, although in most instances the linkage disequilibrium was also weaker among controls (Fig. S5 in the Supplementary Appendix). Areas with darker shading represent stronger linkage disequilibrium. Int denotes intergenic region, and Pr MUC5B promoter.

Figure 3. MUC5B Expression in 33 Subjects with Idiopathic Pulmonary Fibrosis (IPF) and 47 Healthy Controls, Stratified According to MUC5B Promoter Single-Nucleotide Polymorphism (SNP) (rs35705950) Genotype and Smoking Status

Panel A shows the distribution of MUC5B expression among subjects with wild-type or heterozygous rs35705950 genotypes of MUC5B according to the presence or absence of IPF. Panel B shows MUC5B expression among all controls, controls who smoked, and controls who did not smoke, according to rs35705950 genotype. Panel C compares MUC5B expression in all subjects with IPF, those who smoked, and those who did not smoke, according to rs35705950 genotype. In all the panels, the horizontal lines indicate group medians, and the expression of MUC5B is shown in relation to the expression of the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH).

Figure 4. Immunohistochemical Staining of MUC5B in Lung Tissue from Subjects with Idiopathic Pulmonary Fibrosis and Controls

Immunohistochemical staining showed MUC5B distribution in the cytoplasm of the secretory columnar cells of the bronchi and larger proximal bronchioles in a specimen of lung tissue from a control subject (Panel A). In subjects with idiopathic pulmonary fibrosis, regions of dense accumulation of MUC5B were observed in areas of microscopical honeycombing and involved patchy staining of the metaplastic epithelia lining the honeycomb cysts (Panel B). Accumulation was also observed in the mucous plugs within the cysts (Panel C).