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Published ahead of print on December 15, 2005, doi:10.1165/rcmb.2005-0151OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 496-499, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0151OC

The Mucin-1 568 Adenosine to Guanine Polymorphism Influences Serum Krebs von den Lungen-6 levels

Rob Janssen*, Adrian Kruit*, Jan C. Grutters, Henk J. Ruven, Wim B. Gerritsen and Jules M. van den Bosch

Heart Lung Center Utrecht, Department of Pulmonology, and Department of Clinical Chemistry, St. Antonius Hospital, Nieuwegein, The Netherlands

Correspondence and requests for reprints should be addressed to Dr. J. C. Grutters, Department of Pulmonology, St Antonius Hospital, Koekoekslaan 1, 3435 CM Nieuwegein, the Netherlands. E-mail: j.grutters{at}antonius.net


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Krebs von den Lungen (KL)-6 offers a new perspective as a disease marker in pulmonary diseases. The aim of this study was to analyze whether serum KL-6 levels are dependent on the functional adenosine to guanine mucin-1 (MUC1) gene polymorphism at nucleotide position 568 in a well-characterized white population. Polymorphisms were determined in 327 healthy, white individuals and 74 patients with sarcoidosis, using a PCR-sequence–specific primer assay. The serum KL-6 levels were measured by ELISA. Significant differences between serum KL-6 levels of healthy subjects who were grouped according to MUC1 568 genotype were observed (P < 0.0001) (mean ± SEM): AA (195.2 ± 9.9 U/ml; 95% confidence interval [CI], 175.7–214.8), AG (246.0 ± 8.6 U/ml; 95% CI, 229.0–263.1), and GG (302.6 ± 11.8 U/ml; 95%CI, 279.3–326.0). In the patients with sarcoidosis, the results were (mean ± SD): AA (550.1 ± 411.7; 95% CI, 380.2–720.1), AG (716.3 ± 452.4; 95% CI, 547.4–885.2), GG (1,151.0 ± 1122; 95% CI, 610.1–1692.0); P = 0.02. Comparison of the KL-6 levels in which the 568 genotype was ignored rendered 6 out of 74 (7.5%) misclassifications of "elevated" versus "normal" KL-6 levels or vice versa. In conclusion, the MUC1 568 A to G polymorphism may be of interest for diagnostic purposes because our study delivered in vivo evidence that it contributes to interindividual variations in KL-6 levels.

Key Words: KL-6 • Krebs von den Lungen-6 • mucin-1 • polymorphism


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Many acute and chronic lung disorders with variable degrees of pulmonary inflammation and fibrosis are collectively referred to as interstitial lung diseases (ILDs). To evaluate the activity and monitor the course of the ILD, several methods, such as chest roentgenogram, pulmonary function testing, gallium-67 lung scan, and bronchoalveolar lavage, are available. The lung epithelium–specific protein Krebs von den Lungen-6 (KL-6) offers a new perspective as disease marker in ILDs (1). Serum KL-6 is elevated in a majority of patients with ILD and is normal in patients with bacterial pneumonia or in healthy subjects (2). KL-6 levels depend on the number of regenerating type II epithelial cells and the integrity of the alveolar-capillary membrane (3, 4). Because KL-6 is chemotactic for human fibroblasts, this protein may also play a functional role in fibrosis (5). Serum KL-6 levels predict outcome in idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome, and sarcoidosis (6–9).

The KL-6 antibody recognizes a specific sugar chain on the mucin-1 (MUC1) protein (3). There are known variations in the length and structure of the MUC1 protein that result from two known polymorphisms. The variable number of tandem repeats (VNTR) polymorphism present within the coding region codes for a 20-amino-acid motif, resulting in many different alleles that show a bimodal (i.e., small and large) distribution (10). In addition, the MUC1 pre-mRNA uses one of two neighboring splice acceptor sites for exon 2, resulting in a MUC1 protein difference of nine amino acids. The MUC1 splice site recognition is based on an adenosine (A) to guanine (G) single nucleotide polymorphism in exon 2 at nucleotide position 568 (11). Previously, it has been demonstrated that larger MUC1 proteins express more sugar chains on their surface compared with smaller proteins (12).

We hypothesized that the functional MUC1 polymorphisms might contribute to variance in serum KL-6 levels. We analyzed the MUC1 568 A/G polymorphism using an easy-to-perform PCR-sequence–specific primers (PCR-SSPs) assay in a well characterized Dutch white population. The MUC1 568 genotypes were related to serum KL-6 levels to determine a possible gene–protein relationship.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Healthy Control Subjects
Venous blood samples were obtained from 327 ostensibly healthy employees of the St. Antonius Hospital (210 women [39 12.1 yr of age] and 117 men [42 ± 10.4 yr of age]). By completing a questionnaire, these volunteers provided relevant background information, including medication use, ethnicity, and hereditary diseases. The individuals were assessed with a complete history and physical examination. The over-representation of women who participated in this study is explained by the predominantly female workforce at this hospital. Fifty-five individuals (33 women and 22 men) smoked for at least 5 pack-years. Ethnicity of both parents was used as the criterion for assuming Dutch white ethnicity of the subject. Exclusion criteria included known pulmonary disease and non-Dutch white ethnicity. The medical ethical committee of this hospital approved the study, and all subjects gave formal written consent.

Patients
Seventy-four unrelated and randomly selected Dutch white patients with sarcoidosis (43 men, 31 women; mean age ± SD, 39 ± 11.2 yr) were included in the study. In 49 patients, the diagnosis of sarcoidosis was established when clinical findings were supported by histologic evidence and after exclusion of other known causes of granulomatosis in accordance with the consensus of the ATS/ERS/WASOG statement on sarcoidosis (13). One patient presented with radiographic stage 0, 20 patients presented with stage I, 11 patients presented with stage II, 14 patients presented with stage III, and 3 patients presented with stage IV disease. Twenty-five patients presented with the classic Löfgren syndrome of fever, erythema nodosum, bilateral hilar lymphadenopathy, and joint symptoms. The diagnosis in these patients was made without biopsy.

Serum KL-6 Measurements
KL-6 concentrations were measured by an ELISA technique using a specific KL-6 antibody kit (ED046; kindly provided by Eisai Co., Tokyo, Japan) as described previously (14). All samples were run in duplicate, and mean values were used for analysis.

Sequence-Specific Primers and PCR
The biallelic MUC1 568 A/G single nucleotide polymorphism (exon 2; rs4072037) was determined with PCR-SSPs. The reverse SSPs 5'-AGC TTG CAT GAC CAG AAC CC and 5'-AGC TTG CAT GAC CAG AAC CT were used in combination with the consensus forward primer 5'-CTA TGG GCA GAG AGA AGG AG, leading to expected PCR product sizes of 233 bp. The PCR conditions were as previously described (15).

Statistical Analyses
The statistical evaluation of our data was performed using SPSS 11 (SPSS Inc., Chicago, IL) and Graphpad Prism version 4 (Graphpad Software, Inc., San Diego, CA) software packages. Chi-square test was used for categorical variables and multivariate analysis of continuous variables that were normally distributed. The latter analysis was performed using a linear regression model, controlled for sex and smoking as fixed factors and age as a covariate, followed by a post-test for multiple comparisons between groups. Serum KL-6 levels are reported as estimated marginal mean ± SEM and 95% confidence intervals (CI) in U/ml unless otherwise stated. The reference interval of serum KL-6 in each genotype group was calculated by the following formula: mean ± 1.96 SD. Genotype frequencies were tested for Hardy-Weinberg equilibrium. Statistical significance was denoted by a value of P < 0.05 for all tests performed.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
MUC1 Genotypes
The study population was in Hardy-Weinberg equilibrium for the MUC1 568 genotype distribution (P = 0.44). In healthy control subjects, the MUC1 568 allele frequency was 359 (54.9%) for A and 295 (45.1%) for G. The genotype frequency for AA, AG, and GG was 102 (31.2%), 155 (47.4%), and 70 (21.4%), respectively. In patients with sarcoidosis, the allele frequency was 80 (54.0%) for A and 68 (46.0%) for G. The genotype frequencies were 25 (33.8%) AA, 30 (40.5%) AG, and 19 (25.7%) GG. No significant differences in MUC1 568 genotype or allele frequency distributions were found between patients with sarcoidosis and control subjects.

Relationship between MUC1 568 A/G Polymorphism and Serum KL-6 Levels
Healthy control subjects. The mean serum KL-6 ± SD in all healthy individuals was 238.7 ± 101.5 U/ml, and the reference interval was 39.8–437.6 U/ml. Table 1 summarizes the serum KL-6 levels that were categorized according to MUC1 568 A/G genotype. Pronounced differences between serum KL-6 levels of subjects who were grouped according to MUC1 568 genotype were observed: AA (195.2 ± 9.9 U/ml; 95% CI, 175.7–214.8), AG (246.0 ± 8.6 U/ml; 95% CI, 229.0–263.1), and GG (302.6 ± 11.8 U/ml; 95% CI, 279.3–326.0). Serum KL-6 values were found to be significantly different when all three genotype-group serum levels were compared (P < 0.0001). Post-test analysis was performed for genotype-specific KL-6 levels comparisons (P < 0.0001 between all genotype comparisons). Male subjects tended to have slightly higher KL-6 levels (267.9 ± 8.6 U/ml; 95% CI, 251.0–284.8) than female subjects (234.0 ± 6.7 U/ml; 95% CI, 220.8–247.2; P = 0.019). Smoking had no influence on KL-6 levels (P = 0.8). KL-6 levels were codependent on age (P = 0.01), although grouping the individuals in age brackets 10–19 did not reveal profound differences in KL-6 levels (age 10–19: female subjects, 186.9 ± 7.2 U/ml and male subjects, 224.9 ± 0 U/ml; age 20–29: female subjects, 213.1 ± 10.0 U/ml and male subjects, 214.2 ± 10.1 U/ml; age 30–39: female subjects, 242.2 ± 9.0 U/ml and male subjects, 256.5 ± 12.0 U/ml; age 40–49: female subjects, 214.2 ± 9.4 U/ml and male subjects, 251.1 ± 10.0 U/ml; age 50–59: female subjects, 255.9 ± 10.3 U/ml and male subjects, 271.9 ± 16.8 U/ml; and age > 60: female subjects, 208.2 ± 7.9 U/ml and male subjects, 217.9 ± 12.9 U/ml). Figure 1 illustrates the MUC1 genotype-grouped serum KL-6 levels.


Figure 1
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  		Figure 1. Scatterplot illustrating the association between MUC1 568 A/G genotype and serum KL-6 levels (U/ml) in 327 healthy white subjects. Horizontal bars in scatterplots represent estimated marginal mean for each group.

 

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  		TABLE 1. MUC1 GENOTYPE-SPECIFIC SERUM KL-6 LEVELS IN HEALTHY WHITE SUBJECTS (n = 327)*

 
Patients with sarcoidosis. Figure 2 illustrates the influence of the 568 A/G polymorphism on KL-6 levels in patients with sarcoidosis. When the KL-6 levels of the patients were grouped according to the genotype, the results were AA (550.1 ± 411.7; 95% CI, 380.2–720.1), AG (716.3 ± 452.4; 95% CI, 547.4–885.2), and GG (1,151.0 ± 1,122; 95% CI, 610.1–1,692.0; P = 0.02).


Figure 2
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  		Figure 2. Scatterplot illustrating the association between MUC1 568 A/G genotype and serum KL-6 levels (U/ml) in 74 patients with sarcoidosis. Horizontal bars in scatterplots represent mean for each group.

 
KL-6 levels measured in patients with sarcoidosis at presentation were also dependent on radiographic staging. Namely, patients who had presented with stages 0/I and Löfgren syndrome had lower KL-6 levels (548.9 ± 121.0 U/ml; 95% CI, 307.4–790.3) than those with stages II, III, and IV (1,118.5 ± 133.1 U/ml; 95% CI, 852.9–1,384.2; P = 0.0003). When the 568 A/G genotype influence on KL-6 levels in the patients were corrected for radiographic staging, sex, age, and smoking, significant differences in KL-6 levels between patients grouped according to the MUC1 568 A/G genotype were observed as follows: AA (606.5 ± 136.5 U/ml; 95% CI, 334.0–879.0), AG (670.5 ± 133.9 U/ml; 95% CI, 403.1–937.8), and GG (1,224.2 ± 165.3 U/ml; 95% CI, 894.2–1,554.1; P = 0.003) (Table 2). Smoking, sex, or age did not show any significant influence on KL-6 levels (data not shown).


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  		TABLE 2. MUC1 GENOTYPE-SPECIFIC SERUM KL-6 LEVELS IN PATIENTS WITH SARCOIDOSIS (n = 74)*

 
Comparison of the KL-6 levels in which the 568 genotype was ignored rendered 6 out of 74 (7.5%) misclassifications of "elevated" versus "normal" KL-6 levels or vice versa. Specifically, KL-6 levels of 568 AA patients (n = 3) changed from being elevated (> 437.6 U/ml, according to the reference interval, 39.8–437.6 U/ml) to normal (within to the AA genotype-specific reference interval in Table 1) and 568 GG genotyped patients (n = 3) changed from having normal (39.8–437.6 U/ml) to elevated (> upper reference limit of the GG genotype-specific reference interval in Table 1) KL-6 levels (data not shown).


   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This study is the first to describe a MUC1 genotype influence on KL-6. We demonstrated that the 568 A to G polymorphism contributes to interindividual differences in serum KL-6 levels. Serum KL-6 levels were higher in individuals carrying the 568 G allele, with 568 A homozygotes having the lowest levels, 568 G homozygotes having the highest levels, and heterozygotes having intermediate levels, which is compatible with a gene–dose effect. A similar MUC1/KL-6 gene–dose effect was observed in the patients with sarcoidosis, despite the 3-fold increase of the overall KL-6 levels compared with control subjects. In addition, and as observed previously (9), radiographic staging showing parenchymal involvement (stages II and higher) correlated with a significantly higher KL-6 levels compared with patients without parenchymal involvement (stages 0/I and patients with Löfgren syndrome). Discordant classification of KL-6 levels (normal versus elevated) in 7.5% of the patients with evaluated sarcoidosis clearly demonstrates the consequences of the 568 A/G polymorphism on the interpretation of KL-6 levels.

Ligtenberg and colleagues have demonstrated that the MUC1 gene polymorphism at nucleotide position 568 is functional (11). The choice of the MUC1 pre-mRNA splice acceptor site, resulting in a 27-bp difference between the two alleles, is determined by this polymorphic nucleotide. Furthermore, there is a strong linkage disequilibrium between the MUC1 568 A/G and VNTR polymorphism (11, 16). Pratt and colleagues reported that determination of the MUC1 568 A/G using PCR-SSPs is an easier way to establish MUC1 haplotypes than determination of the VNTR (16). Most alleles containing a large number of tandem repeats have a G at nucleotide position 568 and splice to the upstream splice acceptor site, which results in a larger MUC1 protein. Conversely, most alleles containing a small number of repeats have an A at this position and splice to an acceptor site located 27 bp further downstream, which results in a smaller MUC1 protein (11, 16). The tandem repeat unit is rich in serine and threonine. After translation, the MUC1 protein undergoes extensive modification. Sugar chains are joined through O-glycosylation, with serine and threonine residues of the protein backbone (17). Silverman and coworkers demonstrated that mucins with a high number of repeat units have more sugar chains compared with those with fewer tandem repeats (12). Because the monoclonal IgG1 KL-6 antibody recognizes a sugar chain on the MUC1 protein and O-glycosylation of MUC1 is influenced by the primary sequence of peptide core (3), it is conceivable that the larger MUC1 protein encoded by the MUC1 568 G allele expresses more KL-6 on its surface than the smaller MUC1 protein encoded by the 568 A allele.

Serum KL-6 is a promising disease marker in ILDs (2). Serum KL-6 levels are elevated in a majority of patients with a number of ILDs, including IPF, hypersensitivity pneumonitis, interstitial pneumonitis associated with collagen vascular disease, and sarcoidosis (2, 9). The clinical value of KL-6 measurements as a diagnostic test is likely to benefit from genotyping for the MUC1 polymorphism at position 568. There may be additional SNPs in the MUC1 gene that were not identified and evaluated in this study and that could lead to differential functionality of the gene. Like the 568 A/G polymorphism, such quantitative trait loci may contribute to variance in serum KL-6 levels in addition to SNP 568 A/G. Further scrutiny of the coding and promoter regions of MUC1 is needed to map the MUC1 genetic variability and its phenotypic effect on KL-6 levels more completely.

Serum KL-6 is also a prognostic marker in various lung diseases. High serum KL-6 levels were found to predict a resistance to corticosteroid treatment in patients with IPF (6). Kohno and colleagues estimated survival in IPF using the Kaplan-Meier method and showed that patients with serum KL-6 levels above 1,000 U/ml have a significantly worse prognosis (7). In patients with acute respiratory distress syndrome, KL-6 levels were higher in nonsurvivors than survivors (8). Moreover, serum KL-6 levels tended to be associated with pulmonary disease outcome in sarcoidosis (9). Correcting serum KL-6 levels for the MUC1 haplotypes may increase its value as a prognostic marker.

In conclusion, our study is the first to deliver in vivo evidence that the MUC1 568 A to G polymorphism accounts for significant interindividual variations in serum KL-6 levels.


   Acknowledgments
 
The authors thank Jan Broess and Natalie Pot for their excellent technical support, Pieter Zanen for his support with the statistical methodologies, and the Eisai company for kindly providing the KL-6 ELISA kits.


   Footnotes
 
* These authors contributed equally to this study. Back

Originally Published in Press as DOI: 10.1165/rcmb.2005-0151OC on December 15, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form April 24, 2005

Accepted in final form November 24, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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Copyright © 2006 American Thoracic Society.