Original ArticlesCA repeat allele polymorphism in the first intron of the human interferon-γ gene is associated with lung allograft fibrosis
Introduction
Interferon-γ (IFN-γ) was first recognised for its antiviral activity [1], but a great deal of data has since been accumulated which establishes that this multifunctional cytokine plays an important role in modulating almost all phases of the immune response [2]. One critical aspect of IFN-γ activity is its role in inflammatory responses. Damage due to inflammation, mediated by a number of inflammatory cytokines results in graft fibrosis following lung transplantation. IFN-γ, in particular, plays a pivotal role in the inflammatory response. It has been shown that under inflammatory conditions pulmonary interstitial fibroblasts interact with activated infiltrating T lymphocytes via the CD40-CD40L co-stimulatory pathway and promote both fibroblast and T cell activation [3]. If it is extensive, the normal wound repair process can become pathological, resulting in tissue fibrosis. IFN-γ increases collagen synthesis and fibroblast collagen matrix deposition as well as enhancing the transcription of other matrix genes, including fibronectin [4].
Our group and others have identified a series of polymorphisms in the regulatory regions of cytokine genes which correlate with the amount of cytokine produced 5, 6, 7, 8. It has been shown that a single base change in the interleukin-10 (IL-10) and tumour necrosis factor-α (TNF-α) promoter regions can influence the levels of production of these gene products 5, 8. Functional polymorphisms have also been identified in the signal sequence of the TGF-β gene [7]. Polymorphisms in a range of human cytokine genes have been correlated with transplant rejection 9, 10, fibrosis [11] and autoimmunity 12, 13.
Microsatellites can be used as linkage markers and polymorphic dinucleotide repeats occur within genes for several human cytokines, including TNF-α [14], interleukin-4 (IL-4) [15] and IL-10 [16]. Our group has previously described five alleles of the human IFN-γ gene, which differ by the number of CA repeats in the first intron. Sequence analysis has shown that allele #1 corresponds to 11 CA repeats, allele #2 corresponds to 12 repeats and alleles #3–#5 have 13–15 repeats, respectively. In a parallel study high IFN-γ production, defined by in vitro Con A stimulation of peripheral blood leukocytes followed by ELISA, was correlated with the presence of allele #2 [6]. Individuals homozygous for allele #2 were shown to consistently produce significantly more IFN-γ (p = 0.01, Mann-Whittney test) than individuals with other allele combinations.
The aim of this study was to test the hypothesis that graft fibrosis is exacerbated in recipients with the genetic propensity to produce higher amounts of IFN-γ in response to lung allotransplantation.
A group of 82 lung transplant patients was studied. Patients were from the Cardiothoracic Transplant Unit, Wythenshawe University Hospital of South Manchester and had undergone lung transplantation between January 1990 and August 1996. Control samples came from organ donors and healthy volunteers (mainly Caucasian) from Manchester area.
DNA was extracted from whole peripheral blood using ammonium acetate protein precipitation (Dr. David E Barton, personal communication).
The microsatellite region in the first intron of the IFN-γ gene was amplified using two primers CAT-L (GCTGTCATAATAATATTCAGAC) and CAT-R (CGAGCTTTAAAAGATAGTTCC). Amplifications were carried out on a PTC-100 (Genetic Research Instrumentation Ltd, Dunmow, UK) thermal cycler in 30 μl reaction mixtures containing: 50–200 ng test DNA, 50 mM KCl, 10 mM Tris-HCl, 0,1% Triton X-100, 200 μM each dATP, dCTP, dGTP and dTTP (Life Technologies, Paisley, UK), 2.5 mM MgCl2, 1 M Betaine (Sigma, Poole, UK), 0.5 μM each primer and 1 U Taq polymerase (Life Technologies, Paisley, UK). Following an initial denaturation step (5 min at 95°C) samples were subjected to 30 rounds of PCR consisting of 95°C for 30 sec, 56°C for 30 sec and 72°C for 1 min, with a final extension time of 5 min at 72°C.
8 μl of PCR products were mixed with 3 μl of loading buffer (5xTBE, 49% glycerol, 0.1% SDS, 1% bromophenol blue) and were separated on a polyacrylamide gel containing (12% acrylamide/bis acrylamide, 19:1) at 35 mA for 4 hours. Following electrophoresis, gels were stained with ethidium bromide (10 mg/ml), visualised with UV light on a transillluminator and photographed for the subsequent definition and identification of alleles.
Probability values were calculated by χ2 test.
Section snippets
Results
Alleles as they can be visualised on the polyacrylamide gel are shown in Fig. 1. Sequence analysis confirmed the number of CA repeats (Fig. 2). Four alleles were observed in our control group (n = 164) and their frequencies are shown in Table 1. Allele #2 was the most commonly observed in our control group and the genotype distribution exhibited a preponderance of heterozygotes (47% with genotype #2/#3) and homozygotes (21% with genotype #2/#2) containing this allele. Only 8 out of 15 possible
Discussion
In this study we have analysed polymorphism within the human IFN-γ gene from lung transplant patients and control subjects. The IFN-γ gene, normally present as a single copy, has been assigned to chromosome position 12q24.12 [17]. A polymorphism is located in the first intron of the gene and we have previously described five different alleles of the CA repeat microsatellites in the control population [6]. The frequency of the different alleles may vary depending on the populations under
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