We found that polymorphisms in TGFB1 were associated with asthma and atopy in Mexican children. This is the largest study of TGFB1 polymorphisms in asthma and atopy. The T allele of C-509T, the C allele of T869C, and the C allele of rs7258445 increased the risk of asthma; two of these SNPs are functional. Haplotype analysis results are consistent with the single SNP analyses; the haplotype containing the T allele of C-509T, the C allele of T869C, and the C allele of rs7258445 was related to increased risk of asthma. We also found the same three alleles to be positively associated with the degree of atopy, as assessed by the number of positive skin tests.
We examined five SNPs in TGFB1
based on linkage disequilibrium criteria and functional data. Previous association studies of TGFB1
have mainly focused on SNPs in the promoter [G-800A, C-509T] and in exon 1 [T869C (SNP in codon 10), G915C (SNP in codon 25)]. We evaluated tagging SNPs throughout the gene. An efficient way of selecting tagging SNPs without losing power is to give priority to SNPs with the largest number of other SNPs in high linkage disequilibrium (de Bakker et al. 2005
). We covered all the common SNPs (minor allele frequency ≥ 10%) in the promoter region and the exons. We chose C-509T because of strong evidence that it influences TGFB1 function, promoter activity, and circulating TGFB1 level (Awad et al. 1998
; Grainger et al. 1999
; Silverman et al. 2004
). We also included T869C in codon 10, despite an r2
of 0.87 with C-509T in our Mexican population because it is also functional. The T869C T to C substitution leads to an amino acid change from leucine to proline in the signal peptide resulting in increased secretion of TGFB1 protein in vitro
(Dunning et al. 2003
) and increased circulating TGFB1 concentration (Yamada et al. 2001
; Yokota et al. 2000
). In addition, it has been associated with atopic asthma (Mak et al. 2006
) and cystic fibrosis severity (Drumm et al. 2005
). A polymorphism in codon 25 leads to higher levels of TGFB1 production (Awad et al. 1998
), but it was not found in a Hispanic polymorphism discovery set that includes Mexicans (http://snp500cancer.nci.nih.gov
) and thus we did not study it.
We used resequencing data generated from Caucasians and Mexican-Americans to make our selection of tagging SNPs. There is increasing documentation that tagging SNPs selected from Caucasians provide excellent coverage in other non-African populations (Gonzalez-Neira et al. 2006
; Tenesa and Dunlop 2006
). Tenesa et al. demonstrated that power does not depend on which population is used to select tagging SNPs, especially in non-African populations (Tenesa and Dunlop 2006
). Mexicans are predominantly a mix of Caucasian and Native American ancestry with a minimal African component (Choudhry et al. 2006
). Mexicans from Mexico City and Mexican-Americans should be reasonably similar.
We also found suggestive evidence of a positive association between the T allele C-509T and higher degree of atopy. The T allele of C-509T was associated with elevated total serum IgE in two studies (Hobbs et al. 1998
; Nagpal et al. 2005
), and no association was seen in three studies (Buckova et al. 2001
; Meng et al. 2005
; Silverman et al. 2004
). The C allele of the SNP T869C was associated with increased risk of asthma and higher degree of atopy in our study. Mak et al. found that the CC genotype was related to increased risk of atopic asthma (Mak et al. 2006
), consistent with our finding. The C allele of T869C enhances TGFB1 secretion in vitro independent of any effect of the C-509T polymorphism (Dunning et al. 2003
). There are no published results on the relationship between T869C and the degree of atopy.
We found that the rs7258445 C allele was associated with increased risk of asthma and higher degree of atopy in our population. There are no prior published data on this SNP in relation to asthma or atopy. SNP rs7258445 is in an Alu repeat region. Alu-SNPs may harbor potential sites associated with disease (Ng and Xue 2006
) and Alu sequences can influence gene expression (Batzer and Deininger 2002
). However, the association could simply reflect the r2
of 0.49 with C-509T which is clearly functional. Based on our haplotype analysis, the association with this SNP is probably not independent of the association with C-509T.
One other study (Nagpal et al. 2005
) has examined TGFB1
haplotypes in relation to asthma. Nagpal et al. studied the influences of haplotypes of three polymorphisms [(CT)n
repeat, G-800A and C-509T] on TGFB1 serum level and asthma risk in an Indian population (Nagpal et al. 2005
). They found the haplotype 23_G_T, containing the T allele of C-509T, to be associated with higher TGFB1 serum levels and increased asthma risk, whereas 22_G_C, containing the C allele of C-509T, was associated with lower serum levels of TGFB1 and lower risk of asthma. These results are similar to ours where the most common haplotype TCCCC, containing the risk alleles from C-509T, T869C and rs7258445, was associated with statistically significantly increased asthma risk. As expected, the haplotype CTTCC, with the lower risk alleles at these three sites, was inversely associated with asthma in our data.
The associations that we found between TGFB1
polymorphisms and increased risk of asthma and atopy may result from increased TGFB1
gene expression. TGFB1
is overexpressed in airways of asthmatics compared with healthy controls (Minshall et al. 1997
; Ohno et al. 1996
). In particular, the T allele of C-509T is associated with increased gene expression (Silverman et al. 2004
), and the C allele of T869C results in increased TGFB1 secretion (Dunning et al. 2003
). Both C-509T and T869C may be associated with increased asthma risk based on enhanced TGFB1 function.
TGFB1 has been implicated in asthma pathogenesis by various mechanisms (Duvernelle et al. 2003
). Airway remodeling appears to be important in the pathogenesis of childhood asthma. TGFB1 is a potent profibrogenic factor with major involvement in the initiation and persistence of airway remodeling in asthma (Boxall et al. 2006
). Many cells express TGFB1, including fibroblasts, smooth muscle, epithelial and inflammatory cells such as eosinophils, lymphocytes and mast cells (Duvernelle et al. 2003
). Minshall and coworkers demonstrated increased TGFB1
mRNA and protein expression in eosinophils in the airways of asthmatic patients, resulting in subepithelial fibrosis (Minshall et al. 1997
; Ohno et al. 1996
). TGFB1 plays a role in synthesis and deposition of extracellular matrix proteins. TGFB1 can increase collagen and fibronectin production in asthmatic airways which may contribute to fibrosis and remodeling (Burgess et al. 2006
; Coutts et al. 2001
; Nomura et al. 2002
; Romberger et al. 1992
). TGFB1 has been shown to be a critical mediator of lung fibrosis in animal models, and a number of studies have shown that antagonizing TGFB1 prevents the development of tissue fibrosis (Noble 2003
Recent evidence suggests a role for TGFB1 in airway hyperresponsiveness. Specifically, TGFB1 influences airway responsiveness to bradykinin (Bronner 2005
). In human airway smooth muscle cells in culture, TGFB1 has been shown to increase the expression of bradykinin 2 receptors (Kim et al. 2005
) and decrease the number of β-adrenergic receptors (Nogami et al. 1994
). Based on these observations, higher levels of TGFB1 might be related to higher levels of airway responsiveness.
TGFB1 may also influence asthma and atopy etiology via its role in T-cell regulation. TGFB1 blocks Th1 differentiation by means of inhibition of expression of T-bet (T-box expressed in T cells) (Gorelik et al. 2002
). This reduced Th1 differentiation can lead to the increased production of Th2 cytokines such as IL-4 and IL-13, which are important in the allergic asthmatic response to inhaled allergen (Finotto and Glimcher 2004
Our asthma cases were diagnosed by pediatric allergists at a pediatric allergy specialty clinic of a large public hospital. Referral to this pediatric allergy clinic is a tertiary referral, and thus the children in our study had already been seen by a generalist and a pediatrician over time for recurrent asthma symptoms. Diagnoses were made on clinical grounds according to previous guidelines (BTS/SIGN 2003
). We did not have tests of bronchial hyperreactivity. The physician diagnosis of asthma is a valid outcome compared to objective measurements (Jenkins et al. 1996
). Of note, Kauppi et al found that 87% of self-reports of asthma diagnosis among Finnish adults were confirmed by objective data (Kauppi et al. 1998
). Phenotyping errors result in reduction in the power of genetic association studies (Edwards et al. 2005
). The resulting non-differential misclassification may also bias results toward the null. Misclassification with chronic obstructive pulmonary disease, a potential problem in adults, is a concern lacking in children. We had objective data on atopy; skin prick tests revealed that the vast majority of these children with asthma (92%) to be atopic to aeroallergens.
Associations of TGFB1
polymorphisms with asthma and atopy were not observed in all studies. This might reflect differences in study size, asthma classification, or populations. Phenotyping differences can contribute to lack of replication (Zheng and Tian 2005
). We studied allergic asthmatic children, whereas some others studied adult asthma patients. Ours is the largest study (546 cases) after that of Silverman et al. (527 cases) (Silverman et al. 2004
) which found similar results; other studies had 30 to 231 cases and may have been underpowered. In examining reasons for non-replication in genetic association studies, Lohmueller et al. found that small sample size was the major factor (Lohmueller et al. 2003
In summary, we found that three SNPs in TGFB1, including the functional SNPs C-509T and T869C, influence childhood asthma and the degree of atopy in a Mexican population. These data add to increasing mechanistic and epidemiologic evidence for a role of TGFB1 in asthma and atopy.