Abstract
Purpose Mucin glycoproteins contribute to lung pathophysiology in asthma. The protein backbone of mucin glycoproteins is encoded by specific MUC genes, which exhibit a high degree of polymorphisms that generate a variable number of tandem repeat (VNTR) domains. MUC7 typically encodes for 6 VNTRs, each with 23 amino acids. In a northern European cohort, a polymorphism encoding MUC7*5 (5-VNTR) is in 100% linkage disequilibrium with the single nucleotide polymorphism rs9982010 and associated with a decreased risk of being asthmatic and having better lung function. African Americans have a 5- to 10-fold increase in incidence of asthma relative to whites, who are believed to be partially associated with higher genetic susceptibility. Occurrence of the rs9982010 and MUC7 allelic frequencies was evaluated in inner-city African Americans to test their association with a diagnosis of asthma.
Methods Genomic DNA, collected from a cohort of African American asthmatic subjects, was used to detect the MUC7 VNTR polymorphisms and to analyze the rs9982010 single nucleotide polymorphism.
Results A logistic regression analysis showed that the MUC7*5-VNTR allele decreased the likelihood of a diagnosis of asthma (odds ratio, 0.173 [95% confidence interval, 0.041-0.737]; P < 0.018) and is not in a strong linkage disequilibrium with the rs9982010 (r 2 = 0.03; odds ratio, 66; confidence interval, 5.913-736.72). A novel MUC7*4-VNTR polymorphism, identified in an African American nonasthmatic individual, was linked to a structural rearrangement of the VNTR domain.
Conclusions These data extend the association of MUC7*5 allelic polymorphisms and asthma to inner-city African Americans.
Asthma is a complex, multifactorial disease reflecting genetic and environmental components. Asthma is now regarded as having multiple different subtypes rather than being a single disease entity.1The morbidity and mortality associated with asthma are disproportionately high among minority pediatric populations, particularly those who reside in densely populated inner-city areas.2-6African Americans are hospitalized for asthma 3 times more often than other Americans, and African Americans living in inner cities are 2 to 6 times more likely to die of asthma.7Inner-city and social risk factors likely explain some of the disparities in incidence; however, genotype is an important determinant of host immune responses and contributes to the overall predisposition of individuals to asthma. The contributions of genetic background to the development of asthma in minority populations are understudied.
Mucin glycoproteins (mucins) are the major macromolecular components of the lung mucous layer, which protects the respiratory tract epithelium against infectious agents, allergens, and environmental toxins. Mucins are overproduced in asthma and other lung diseases and contribute to airway pathophysiology and thus to disease morbidity and mortality.8-12 MUC genes encode the protein backbone of mucins, and most of the MUC genes have polymorphisms that encode a variable number of tandem repeats (VNTRs)13(reviewed by Rose and Voynow12) that can result in differences in the length of the protein backbone. Genetic analyses of some MUC genes have been carried out in patients with atopy and/or asthma. A longer VNTR length in the MUC2 gene is associated with a cohort of atopic, nonasthmatic patients, but no associated differences with asthma and VNTR domains of MUC1, MUC4, MUC5AC, or MUC5B genes have been found.14
However, a study using a northern European cohort showed an association between the risk of having a diagnosis of asthma and a polymorphism in MUC7.15Typically, MUC7 encodes 6 nonperfect VNTRs (MUC7*6) of 23 amino acids, with the less common MUC7 polymorphic variant containing 5 VNTRs (MUC7*5).16A study by Kirkbride et al.15in a northern European cohort identified an association of the MUC7*5 allele with a decreased risk of an asthma diagnosis. A subsequent study identified the rs998210 single nucleotide polymorphism (SNP) in 100% linkage disequilibrium (LD) with the MUC7*5 polymorphism in the same northern European population.17Because African Americans have a higher prevalence of asthma, we hypothesized that they would also have a lower prevalence of the apparently protective MUC7*5 allele. We therefore investigated the MUC7 VNTR domain and the occurrence of the rs998210 SNP in the MUC7 gene to determine its association with asthma in inner-city African American subjects. These 2 polymorphisms were focused on because they are the only MUC7 polymorphisms that have been associated with asthma to date.
MATERIALS AND METHODS
Human Subjects
The Asthma Severity Modifying Polymorphisms (AsthMaP) project provided asthmatic patient samples for our study. The AsthMaP is a study of gene-environment interactions in inner-city pediatric asthma patients treated at Children's National Medical Center, Washington, DC. The nonasthmatic controls were adolescents selected from an ongoing genetic study at Children's National Medical Center on metabolic syndrome in inner-city adolescents.
DNA Isolation
Whole blood or buccal swab samples were collected from asthmatic individuals and nonasthmatic controls under an institutional review board-approved protocol. Genomic DNA was isolated by standard protocols.
MUC7 VNTR Polymorphism Genotyping
The approach used by Rousseau et al.17was used to evaluate the VNTR polymorphisms in the genomic MUC7 gene for each subject. Briefly, polymerase chain reaction (PCR) amplification of genomic DNA was carried out using primers designed to span the entire VNTR domain. The location of the primers is indicated by arrows in Figure 1. The sense primer 5′-cagaatgccaccaccatatcttcaa-3′ and the antisense primer 5′-ggtgcaagagtagttggggaagaat-3′ are located at nucleotides 400 to 425 and 959 to 984, respectively, on the genomic MUC7 DNA in exon 3 (chr 4q13-q21; accession number, L13283).
DNA Sequencing
Polymerase chain reaction products were electrophoresed on a 2% ethidium bromide agarose gel and visualized on a Chemidoc Imager (BioRad, Hercules, CA). Bands identified for DNA sequencing were excised and extracted using QiaQuick gel extraction kit (Qiagen, Valencia, CA), ligated into pCRII-TOPO (Invitrogen, Carlsbad, CA), and sequenced (Davis Sequencing, Davis, CA).
Single Nucleotide Polymorphism Analysis
TaqMan SNP Genotyping Assays analysis of the rs998210 SNP was carried out using specifically designed kits (Applied Biosystems, Foster City, CA) on an ABI 7900HT TaqMan machine. The SNP genotyping assay targeted to MUC7 determined the C/T transition, located at chr 4-71380925.
Statistical Analysis
The frequency of each MUC7 polymorphism was imported into a contingency table (Table 1). A χ2 test was then used to statistically evaluate whether there was an association between the MUC7 polymorphisms and asthma. To evaluate the associated risk of an asthma diagnosis and the MUC7 allelic polymorphisms in an African American cohort, logistic regression models (Stata v10; StataCorp, College Station, TX) were used to generate relative odds ratios (ORs) and 95% confidence intervals (CIs). These analyses were repeated to determine the association of the MUC7*5 polymorphism and the rs998210 SNP in the same population. Hardy-Weinberg equilibrium was tested for each SNP using a 1-degree of freedom χ2 test.
Because of the limited availability of a control population, a post hoc power analysis was performed to determine the likelihood of finding a significant difference.
RESULTS
Demographics
The sample population was limited by the enrollment of the pediatric population in the AsthMaP study and other non-airway related studies. Our population comprised 84 inner-city patients in the AsthMaP study and 37 nonasthmatic controls. The age and sex of the cohort are reported in Table 2. Overall, the sample population was 46% male and 57% asthmatic patients.
The post hoc power analysis comparing the TR polymorphism in asthmatic patients and nonasthmatic controls, whose sample sizes were of 84 and 37 respectively, showed that we had a 66% power to detect a significant difference (P < 0.05).
Allelic Variation in the Number of VNTR Domains
The 2 previously reported MUC7 VNTR polymorphisms, MUC7*6 and MUC7*5, were identified in our study. They were observed as either homozygous 6/6 (Fig. 2, lane 1: 559 base pairs [bp]) or heterozygous 6/5 (Fig. 2, lane 2: 490 bp) allelic pairings.
We also identified, in a control subject, what looked like a novel polymorphism predicted to encode 4 VNTRs (Fig. 2, lane 3: 421 bp), as the amplicon corresponded to 69 bp (size of a single TR) less than MUC7*5. To assess this, DNA from the PCR product (Fig. 2) was sequenced and shown to encode 4 VNTRs. The first encoded VNTR repeat domain contained 2 SNPs that altered the genotype and resulted in changes in the amino acid sequence (Fig. 3). The SNPs resulted in a P169T and a T176S change, indicated in Figure 3 as TR1Δ2 with highlighted changes. Unlike MUC7*5, MUC7*4 showed a rearrangement of the order of its VNTR domains as TR1Δ2 followed by TR2, TR1, and TR2 (Fig. 3). This previously unidentified MUC7 VNTR is now designated MUC7*4.
Frequency Analysis of the MUC7*6 and MUC7*5 Allelic Polymorphisms
DNA samples from nonasthmatic and asthmatic patients (Table 2) were analyzed for the frequency of each polymorphism. The frequencies identified in the asthmatic population were MUC7*6 allele, 0.99 and MUC7*5 allele, 0.01. In the control population, the frequency results were MUC7*6 allele, 0.91; MUC7*5 allele, 0.08; and MUC7*4 allele, 0.01 (Table 1).
We evaluated the expression of MUC7 allelic polymorphisms and the associated risk of having a diagnosis of asthma both in the AsthMaP and control cohorts. A logistic regression analysis of the association of MUC7*5 allelic polymorphism and not having a diagnosis of asthma gave an OR of 0.173 with a 95% CI of 0.041-0.737 and a P value of 0.018 (Table 3). These data were supported by a χ2 analysis showing a significant association of MUC7*5 (P = 0.01) with not having a diagnosis of asthma as a child.
Single Nucleotide Polymorphism Analysis of rs998210
The previously identified SNP rs9982010 in the second intron of the MUC7 gene was earlier shown to be in 100% LD with the MUC7*5 allelic polymorphism in a northern European cohort.17This SNP was also analyzed in our African American cohort. Our data showed that with the T-to-C conversion, there is a low LD between the C/T SNP and the MUC7*5 allelic polymorphism. These data show that the African American population only showed an LD measured by r 2= 0.03. These findings are not in concordance with the results in the northern European cohort where a 100% LD with the polymorphic allele is observed. Both the MUC7*5 and the SNP were in Hardy-Weinberg equilibrium (MUC7*5 polymorphism, P = 0.12; rs998210, P = 0.29). A logistic regression analysis performed between the MUC7*5 allelic polymorphism and the rs9982010 SNP showed an OR of 66.0 (P < 0.0001; 95% CI, 5.913-736.72), indicating that the African American individuals with the MUC7*5 allelic polymorphism were 66 times more likely to have the T-to-C conversion in rs9982010, irrespective of asthma.
DISCUSSION
African Americans have an increased incidence of asthma and are 3 times more likely to be hospitalized with asthma-related symptoms. In addition, African Americans living in inner cities are 2 to 6 times more likely to die of asthma.7,18An individual's overall genotype and environment can predispose one to asthma, but the contributions of genetic background to the risk of having a diagnosis of asthma are uncertain.
Mucin overproduction is implicated in asthma.12Thus, an association of MUC genes with asthma was carried out by Swallow and co-workers. The data showed no association of VNTR numbers in allelic variants in the MUC1, MUC2, MUC4, MUC5AC, and MUC5B genes.14However, this group identified a polymorphism in the MUC7 gene, MUC7*5, with an allelic frequency of 0.10 associated with a decreased risk of having a diagnosis of asthma in a northern European asthmatic cohort.15This data suggested that the MUC7*5 allele was protective against asthma, and a subsequent longitudinal study supported the concept that the MUC7*5 allele had a protective effect on respiratory function.
Because minority pediatric populations in inner cities have a higher prevalence of asthma, we predicted that our cohort would have a lower prevalence of the MUC7*5 allele if it were protective. Our data showed an allelic frequency of 0.05, which is lower than that observed (0.1) for the MUC7*5 allele in a northern European cohort.15Interestingly, this is supported by a small African cohort (n = 29) that also shows a reduction in the MUC7*5 allele frequency15of 0.052. Although each of the 2 sample sets-northern European cohort and inner-city African Americans-are relatively small, the combined data support the hypothesis of a reduction in the frequency of the protective MUC7*5 allelic polymorphism in a population that is of greater risk of having a diagnosis of asthma.
In the northern European asthmatic cohort, the rs998210 intronic SNP in the MUC7 gene has been shown to be in 100% LD17with the MUC7*5. Our data showed that this SNP is significantly associated with the MUC7*5 polymorphism (P = 0.0007) in African Americans, but not at 100% LD. Rousseau et al.17suggested that this SNP might not be functional, as it does not reside in an identified motif region. These data on MUC7 allelic polymorphisms highlight one example where a small genetic difference between ethnically diverse populations could impact the susceptibility of having a diagnosis of asthma, especially in a high-risk inner-city population.
Although MUC7*6 and MUC7*5 seem to be the 2 most predominant alleles in the human population, unique MUC7 alleles have been identified, for example, MUC7*8 in a northern European cohort with atopic asthma.15Herein, we identified a novel MUC7*4 polymorphism in an African American nonasthmatic individual that resulted in a reduction in the number and rearrangement of the encoded TR domains. Two SNPs within the first TR resulted in a change in the amino acid sequence of the first encoded TR (TR1Δ2). The VNTR domains of MUC7*4 is TR1Δ2, TR2, TR1, and TR2, in contrast to TR1 to 6 of MUC7*6 and TR1, 2, 3, 5, and 6 of MUC7*5.
The role of mucins in the mucosal immune system is only beginning to be understood at the molecular level.19Mucins are overproduced in acute and chronic airway diseases and contribute to the disease morbidity and mortality. MUC7 is a small secreted mucin glycoprotein (180 kd) expressed predominantly in the submandibular and sublingual glands20and salivary secretions.21,22MUC7 has been shown to bind to bacteria, and small recombinant MUC7 peptides exhibit antibacterial, antifungal,22-24and antiviral properties.25We have recently shown that MUC7 mucin is present in the airway secretions of asthmatic, but not control, pediatric patients,26suggesting that MUC7 mucin may have a role in the pathophysiology of asthma. MUC7, like all mucins, is highly O-glycosylated, and alterations in the sequence and number of encoded VNTR domains could have a significant impact on its biochemical properties and biological functions and thus its role in diseases. Future studies will be needed to determine mechanisms by which polymorphisms in the MUC7 gene alter the host innate immune response of MUC7 mucin and its relevance to asthma.
ACKNOWLEDGMENTS
The authors thank Dr. Anamaris Colberg-Poley and Dr. Joseph Devaney for excellent technical advice and Dr. Eric Hoffman for his insight and careful analysis of this manuscript. Alan Watson was a predoctoral student in the Immunology Program of the Institute for Biomedical Sciences at the George Washington University. This work is from a dissertation presented to the aforementioned program in partial fulfillment of the requirements for the PhD degree.