Association of asthma and transforming growth

factor-β1 polymorphism in children

Wen-Dong Liu, Ji-Rong Lu, Wen-Bin Liu, Kai-Shu Zhao, Cai-Xia Wang and Hong Yu

Qingdao, China

Author Affiliations: Department of Pediatrics, Qingdao Municipal Hospital, Qingdao 266011 (Liu WD, Wang CX and Yu H); Department of Pediatrics, the First Hospital affiliated to Jilin University, Changchun 130021 (Lu JR and Zhao KS); and Jiaozhou Municipal People’s Hospital, Jiaozhou 266300 (Liu WB), China

Corresponding Author: Wen-Dong Liu, MD, Department of Pediatrics, Qingdao Municipal Hospital, Qingdao 266011, China (Tel: 86-532-2789387; Email: liuwd1226@sohu.com)

Background: There are few reports on the relationship of transforming growth factor-β1 (TGF-β1) and asthma. This study was undertaken to explore the association of TGF-β1 gene polymorphism with asthma in children.

Mothods: Ninety-eight children with asthma and 52 normal children were enrolled. The TGF-β1 gene -509C/T polymorphism in the 5’-flanking region was detected using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The total serum IgE level was examined by the sandwich ELISA method.

Results: No significant differences were found in genotype distribution and allele frequencies between asthmatic individuals and normal controls. But significant differences were seen among severe asthma, mild asthma and normal controls. The serum IgE levels of TT genotype in asthmatic individuals were higher than those of CC or CT genetype.

Conclusions: TGF-β1-509C/T gene mutation is not a high risk factor of asthma, but it is closely correlated with asthma severity, and could be one of candidate genes in severe asthma. The level of serum total IgE is related to TGF-β1-509C/T gene mutation homozygotes.

Key words: bronchial asthma; TGF-β1 gene; polymorphism PCR-RFLP; children

World J Pediatr 2005;2:113-116

Introduction

Asthma is a polygenic disease with increasing prevalence and mortality in recent years. Its pathogenesis is a complex one, relevant to factors of heredity, environment, behavior, psychology and immunity. The levels of TGF-β1 are increased in the bronchoalveolar lavage fluid of asthmatics as compared with those of nonasthmatic individuals, and the levels increased after allergen challenge.[1-4] TGF-β1 plays an important role in etiopathogenesis of asthma, which has attracted the attention of many scholars in recent years. This study was designed to detect TGF-β1, C-509T polymorphism by polymerse chain reaction-restriction fragment length polymorphism (PCR-RFLP), and to explore the association of TGFβ and asthma, so as to provide a scientific basis for the prevention and control of the disease.

Methods

Subjects and specimens

Asthmatic group

A total of 98 asthmatic children who had been diagnosed and treated at our hospital according to the criteria for Pediatric Asthma Prevention and Treatment were enrolled.[5] They were divided into mild asthmatics (62 patients) and severe ones (36) according to their clinical manifestations and PEF values. They had not been given any corticosteroids and immune regulators 2 weeks ago.

Control group

Fifty-two normal children were selected randomly, who had been free from respiratory diseases and infections or any personal and family history of other diseases.

Collection of specimens

Two ml venous blood was taken, and 0.5 ml of it was anti-coagulated with 2% EDTA. Chromosome DNA was extracted from whole blood by using genome DNA extraction kit and stored at -20ºC. Another 1.5 ml was centrifuged at 2500 rpm at 4ºC to isolate sera, which were stored at -20ºC for the measurement of total IgE levels.

Methods

Measurement of IgE levels

IgE levels were measured by sandwich ELISA.

TGF-β1-509T polymorphism

In amplification of TGF-β1, gene fragments,[6] primers were synthesized by Beijing Saibaisheng Company, Beijing. Primer sequence: S1, 5’-GGG GAC ACC ATC TAC AGT G-3’, S2, 5’-GGA GGA GGG GGC AAC AGG-3’. Amplification reactions were performed in a volume of 50 μl, containing 100 ng genomic DNA, 20 pmol each primer, 10 mmol Tris-HCl (pH 8.3), 50 mmol KCl, 1.5 mmol MgCl2, 200 μmol dNTP, and 1 U Taq DNA polymerase (Promega Company, USA). Cycling conditions were as follows: PCR reactions were initially denatured at 94ºC for 5 minutes, followed by 35 cycles of denaturation (94ºC for 30 seconds), 30 seconds of annealing at 60ºC, and 30 seconds of extension at 72ºC, and final extension at 72ºC for 5 minutes. PCR contamination was checked by inclusion of negative controls. In genotyping of the TGF-β1 gene by RFLP, amplification products were confirmed by restriction enzyme Eco81I. The products were incubated for 2 hours at 37ºC, analyzed by 3% agarose gelectrophoresis, and visualized after ethidium bromide staining.

Statistical analysis

Genotype distribution frequencies and allele frequencies were analyzed using the chi-square test. The data were expressed as mean±SD. The relation of TGF-β1 (C-509-T) genotypes to serum total IgE level in asthmatic children was studied by analysis of variance and the q test.

Results

PCR reaction products

The expected size of amplification products was 455 bp (Fig. 1). The enzyme digestion showed that the pattern for homozygotes wild type (lane 3), heterozygotes (lane 5) and homozygotes for the base exchange (lane 4) (Fig. 2).

-509C/T gene polymorphism

No significant differences were observed in genotype frequencies and T allele frequencies between the asthmatics and controls (χ2=3.562, P=0.169; χ2=3.607, P=0.058).

TGF-β1 genotype distribution, allele frequency

Significant differences were seen in genotype distribution frequencies and T allele frequencies among mild asthma group, severe asthma group and normal controls (χ2=11.185, P=0.025). A significant difference was detected for this variant when the mild asthma group alone was compared to the severe group (χ2=6.890, P=0.032). The difference was even more significant when the genotypes of the severe asthma group and control group were compared (χ2=9.466, P=0.009). There were significant differences in T allele frequencies between the severe asthma group and the mild asthma group (χ₄²=7.884, P=0.005) and between the severe asthma group and the control group (χ₅²=10.261, P=0.001). However, no significant difference was observed between the mild asthma group and control group (χ₆²=0.337, P=0.562).

Serum total IgE level

The levels of serum total IgE in asthmatic subjects were higher than those of controls, and a significant difference was noted between them. The IgE level of TT genotype asthmatics was higher than CC and CT genotype ones (q1=15.97, q3=13.38, P<0.01), but there was no significant difference between CC and CT genotypes (q2=2.74, P>0.05).

 Table 2. The association of TGF-β1 (C-509T) genotype distribution, allele frequency with asthma severity

 Table 3. The correlation of TGF-β1 (C–509-T) genotypes and serum total IgE level in asthmatic children

Discussion

The pathogenesis of asthma is complicated by a variety of genetic and environment factors. As a polygenic disorder, searching a candidate gene in asthmatic molecular genetics has become a hot topic. In this chronic inflammatory disease of the airways, several cytokines forming a network participate in the activation and infiltration of eosinophils and the expression of the products. However, TGF-β1 is very important in this network.[7-10] The human TGF-β1 gene on chromosome 19q13[11] contains six and seven eons. The promoter for TGF-β1 contains two major sites for the initiation of transcription and multiple regulatory motits. Polymorphisms in promoter sequence of genes result in abnormal transcriptional regulation and thereby influence the severity of the disease. The expression of TGF-β1 is influenced by polymorphisms in the TGF-β1 gene, and some of these polymorphisms may be associated with asthma and other diseases.[12-15] -509 bp position is one of them, and its study is very important to understand asthma.

Seven TGF-β1 polymorphisms have been reported elsewhere.[16] Three of these allelic variations were localized in the 5’-flanking region of the TGF-β1 gene (at positions -98C/A, 800A/G, and -509C/T), 3 were in the coding region (+869T/C, +915G/C), and 263, and an insertion in the 5’-untranslated region at position +72. These findings show that only -509C/T variant is the most informative marker of TGF-β1 contribution to asthma. It is associated with severe asthma[17] and elevated total serum IgE levels.[18] In our study, the TGF-β1 gene C-509T mutation had three genotypes (CC, CT, TT) at position -509. There was a great proportion of individuals with TT genotype in the severe group compared to the mild asthmatics and the controls; however, there was no significant difference between the mild asthmatics and the controls. It is suggested that C-509T variant is associated with asthma severity. Analysis of the total serum IgE levels of different genotypes in asthmatic children revealed that the IgE level of TT genotype asthmatics was higher than that of CC and TT genotype ones; but there was no significant difference between CC and CT genotypes, suggesting that variant C-509T was related to elevated IgE levels. The result was identical to Hobbs’ result, but Bockova et al[19] and Silverman[20] confirmed that C-509T polymorphism is not correlated with total serum IgE. Possibly it is related to sex, age, race, generation, and environment.

The cause of TGF-β1 variation is obscure, but three explanations may address the relation of -509T to asthma severity. First, TGF-β1-509 is a functional variant, located in the promoter region of TGF-β1 and it may destroy a transcription factor  binding site, thus leading to TGF-β1 abnormal expression. Second, -509T association is characterized by the variant in linkage disequilibrium with the true functional polymorphism. Third, TGF-β1 contribution to asthma severity may comprise the interaction of two or more polymorphisms linked to haplotype I. This phenomenon has been identified in the 2-adrenergic receptor gene, where associations can be found between bronchodilator response to agonist in asthmatics and haplotype pairs, but not individual SNPS.[21] It is possible that elevated serum IgE level may be influenced by TGF-β1 inhibiting proliferation of mast cells and preventing IgE synthesis, while eliminating hematopoietins for eosinophils to induce apoptosis.[22]

As a polygenetic disease with complicated characteristics, the heritability of asthma is as high as 70%-80%.[23] Since TGF-β1 gene -509C-T polymorphism is correlated with the severity of the disease, it can be a candidate gene, with which it is good to screen high risk cohort of asthma patients. Therefore, early diagnosis according to the results of research will be effective in prevention and treatment of asthma.

Ethical approval: Not needed.

Competing interest: None declared.

Contributors: LWD wrote the first draft of this paper. All authors contributed to the intellectual content and approved the final version. LJR is the guarantor.

References

1   Redington AE, Madden J, Frew AJ, Djukanovic R, Roche WR, Holgate ST, et al. Transforming growth factor-β1 in asthma: measurementin bronchoalveolar lavage fluid. Am J Respir Crit Care Med 1997;156:642-647.

2   Minshall EM, Leung DY, Martin RJ, Song YL, Cameron L, Ernst P, et al. Eosinophil-associated TGF-β1 mRNA expression and airways fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1997;17:326-333.

3   Ohno I, Nitta Y, Yamauchi K, Hoshi H, Honma M, Woolley K, et al. Transforming growth factor -β1 (TGF-β1) gene expression by eosinophils in asthmatic airway inflammation. Am J Respir Cell Mol Biol 1996;15:404-409.

4   Tillie-Leblond I, Pugin J, Marquette CH, Lamblin C, Saulnier F, Brichet A, et al. Balance between proinflammatory cytokines and their inhibitors in bronchial lavage from patients with status asthmaticus. Am J Respir Crit Care Med 1999;159:487-494.

5   Chinese pediatric asthma prevention and cure cooperation group. Pediatric asthma prevention and cure routine. Chin J Pediatr 1998;36:747-750.

6   Lario S, Inigo P, Campistol JM, Poch E, Rivera F, Oppenheimer F. Restriction enzyme-based method for transforming growth factor-β1 genotyping: nonisotopic detection of polymorphisms in codons 10 and 25 and the 5’-flanking region. Clin Chem 1999;45:1290-1292.

7   Borish L, Rosenwasser LJ. Update on cytokines. J Allergy Clin Immunol 1996;97:719-734.

8   Hansen G, McIntire JJ, Yeung VP, Berry G, Thorbecke GJ, Chen L, et al. CD4 T helper cells engineered to produce latent TGF-β1 reverse allergen-induced airway hyperreactivity and inflammation. J Clin Invest 2000;105:61-70.

9   Nakao A. Is TGF-β1 the key to suppression of human asthma? Trends Immunol 2001;22:115-118.

10 Sime PJ, Xing Z, Graham FL, Csaky KG, Gauldie J. Adenovectormediated gene transfer of active transforming growth factor-β1 induces prolonged severe fibrosis in rat lung. J Clin Invest 1997;100:768-776.

11  Derynck R, Rhee L, Chen EY, Van Tilburg A. Intron-exon structure of the human transforming growth factor-β precursor gene. Nucleic Acids Res 1987;15:3187-3189.

12 Grainger DJ, Heathcote K, Chiano M, Snieder H, Kemp PR, Metcalfe JC, et al. Genetic control of the circulating concentration of transforming growth factor type β1. Hum Mol Genet 1999;8:93-97.

13 Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV. Genotypic variation in the transforming growth factor-β1 gene: association with transforming growth factor-β1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation 1998;66:1014-1020.

14 Watanabe Y, Kinoshita A, Yamada T, Ohta T, Kishino T, Matsumoto N, et al. A catalog of 106 singlenucleotide polymorphisms (SNPs) and 11 other types of variations in genes for transforming growth factor-β1 and its signaling pathway. J Hum Genet 2002;47:478-483.

15 Xaubet A, Marin-Arguedas A, Lario S, Ancochea J, Morell F, Ruiz- Manzano J, et al. Transforming growth factor-β1 gene polymorphisms are associated with disease progression in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2003;168:431-435.

16 Cambien F, Ricard S, Troesch A, Mallet C, Generenaz L, Evans A, et al. Polymorphisms of the transforming growth factor-β1 gene in relation to myocardial infarction and blood pressure. The Etude Cas-Temoin de l’Infarctus du Myocarde (ECTIM) Study. Hypertension 1996;28:881-887.

17 Pulleyn LJ, Newton R, Adcock IM, Barnes PJ. TGF-β1 allele association with asthma severity. Hum Genet 2001;109:623-627.

18 Hobbs K, Negri J, Klinnert M, Rosenwasser LJ, Borish L. Interleukio-10 and transforing growth factor-β promoter polymorphism in allergies and asthma. Am J Respir Crit Care Med 1998;158:1958-1962.

19 Buckova D, Izakovicova Holla L, Benes P, Znojil V, Vacha J. TGF-β1 gene polymorphisms. Allergy 2001;56:1236-1237.

20 Silverman ES, Palmer LJ, Subramaniam V, Hallock A, Mathew S, Vallone J, et al. Transforming growth factor β1 promoter polymorphism C-509T is associated with asthma. Am J Respir Crit Care Med 2004;169:214-219.

21 Drysdale CM, McGraw DW, Stack CB, Stephens JC, Judson RS, Nandabalan K, et al. Complex promoter and coding region β2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci USA 2000;97:10483-10488.

22 Alam R, Forsythe P, Stafford S, Fukuda Y. Transforing growth factor-β abrogates the effects of hematopoietins on eosinophils and induces their apoptosia. J Exp Med 1994;179:1041-1045.

23 Yi XJ, Li WM. The genetic progress of children asthma. Chin J Pediatr 1999;37:770-771.

Received April 26, 2005; Accepted after revision June 22, 2005