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Division of Medical Science, University of Birmingham, Queen Elizabeth Hospital (J.M.H., R.N., A.A., S.G., J.A.F., S.C.L.G.), Birmingham B15 2TT; and Birmingham Heartlands Hospital, (S.C.L.G.), Birmingham B9 5SS, United Kingdom
Abstract
Graves’ disease (GD) is an autoimmune thyroid disease of unknown etiology, although predisposition to the development of this disease is thought to be caused by both genetic and environmental factors. Recently, an association between a promoter polymorphism of the interleukin 4 gene and GD has been reported. A C-T base change at position -590 showed modest protection against the development of GD in a United Kingdom data set of 135 patients with GD and 101 controls. This polymorphism was, therefore, investigated in a much larger case-control cohort of 384 patients with GD and 288 control subjects using PCR, followed by restriction fragment length polymorphism analysis. No protective effect of the T allele of this polymorphism was observed in our data set, and indeed no significant difference in either allelic or genotypic distribution was seen between the patient and control groups. Moreover, calculation of probabilities indicate that the original study lacked sufficient power to support the conclusions drawn. Our data support the hypothesis that the C-T promoter polymorphism of the interleukin 4 gene does not confer protection against the development of GD in Caucasians in the United Kingdom.
GRAVES’ DISEASE (GD) is an autoimmune disease of the thyroid gland characterized by hyperthyroidism, diffuse goiter, and positive thyroid autoantibodies. The etiology of the disease is unknown, but its development is likely to be influenced by multiple protective and permissive environments in genetically susceptible individuals. Evidence for a genetic effect comes from family and twin studies (1). The candidate gene approach, in both family and case control data sets, has identified that both the human leucocyte antigen region on chromosome 6p21 and the CTLA-4 gene region on chromosome 2q33 loci confer susceptibility to GD. A genome-wide search has revealed three additional regions conferring susceptibility to GD: GD1, GD2, and GD3 on chromosomes 14q31, 20q11, and X, respectively; and linkage has also been reported on chromosome 18q21 (1).
A recent study by Hunt et al. (2), from a data set of 135 patients with GD and 101 controls, has suggested a modest protective effect for the T allele of a promoter polymorphism (at position -590) in the interleukin-4 (IL-4) gene. Replication of susceptibility loci in additional data sets is a prerequisite to the further study of how candidate genes may lead to the development of the autoimmune disease process and GD. The aim of this study was, therefore, to determine whether the -590 C/T polymorphism of the IL-4 gene was associated with GD in our large case-control cohort.
Subjects and Methods
Caucasian patients (with both parents and both grandparents of British or Irish origin) with GD were recruited from four large thyroid clinics in Birmingham, Bournemouth, and Exeter, United Kingdom. Patients with GD were defined by the presence of biochemical hyperthyroidism, together with two of the following criteria: diffuse goiter; a significant titer of thyroid peroxidase, thyroglobulin, or TSH receptor autoantibodies; and the presence of dysthyroid eye disease. Thyroid peroxidase and thyroglobulin autoantibodies were measured by gelatin particle agglutination (SERODIA-AMC and SERODIA-ATG, Fujirebio Inc., Tokyo, Japan), and a titer of 1:100 was considered significant for both assays. TSH receptor autoantibody status was determined by a radioactive inhibition method (RSR Ltd., Cardiff, UK). A value of 10 U/liter or more was deemed significant after comparison with results from 50 control sera obtained from the local Blood Transfusion Service. In total, DNA was obtained from 384 index cases with GD. Ethnically matched control subjects with no history of autoimmune disease were bled at various sites, including the Blood Transfusion Service, Birmingham Heartlands Hospital, and the Queen Elizabeth Hospital, Birmingham. In total, DNA was obtained from 288 control subjects. All controls were biochemically euthyroid, with insignificant thyroid autoantibody titers. This study was approved by the respective local ethics committees, and all subjects gave informed written consent.
DNA was extracted from whole blood using the Nucleon Bacc II kit (Nucleon Biosciences, Coatbridge, Lanarkshire, UK). Amplification of the target DNA in the promoter region of the IL-4 gene was performed by PCR using primers AW41A and AW41B, as described previously (3). The reaction was carried out in a final volume of 25 µl containing 40ng genomic DNA, 50 ng/µl of each primer, 200 µM/liter dNTPs, 1 mM magnesium chloride, and 1U Taq DNA polymerase (Bioline, London, UK) with appropriate buffer. Amplification was performed in an MJ Research, Inc. Tetrad thermal cycler with an initial denaturation of 94 C for 10 min, followed by 32 cycles of 94 C for 30 sec, 57 C for 30 sec, and 72 C for 30 sec. A final extension step of 72 C for 10 min was performed at the end of the cycling step. This produced an amplicon of 252 bp, which was visualized on a 2% ethidium bromide-stained agarose gel. Restriction fragment length polymorphism analysis of the -590 C/T polymorphism was performed on 5 µl PCR product using 1 U BsmFI (New England Biolabs, Inc., Hitchin, Hertfordshire, UK) enzyme with appropriate buffer at 65 C for 2 h. This yielded two fragments of 192 and 60 bp in the presence of the wild-type sequence and a single fragment of 252 bp in the presence of the mutation. These products were visualized on a 2% ethidium bromide-stained agarose gel.
The allele and genotype frequencies between patients and
control subjects were analyzed by the 
2 test
statistic using Minitab 12 software. A P value of
less than 0.05 was considered to be significant. Estimations of power
and sample size were made using a two-sample test of proportions and
standard probability tables (4).
Results
Unambiguous genotypes for the -590 C/T promoter polymorphism in the IL-4 gene were obtained for 381 of 384 patients with GD and 285 of 288 control subjects available for study.
Table 1
summarizes the allele
frequencies in patients with GD and control subjects. No significant
difference in allele frequency was observed between the two groups.
Specifically, no reduction in the frequency of the T allele was
observed in the patients with GD (14.3%) compared with controls
(12%).
|
summarizes the genotype
frequencies in patients with GD and control subjects. No significant
difference was observed in genotype frequency between the two groups.
Specifically there was no evidence for a reduction in the CT genotype
in patients with GD (26%) compared with controls (20.3%).
|
The recent study by Hunt et al. (2) suggested that the T allele of the -590 C/T polymorphism in the promoter region of the IL-4 gene, or a gene in linkage disequilibrium with this allele, may confer modest protection against the development of GD in the United Kingdom. This was attributed to a decreased frequency of CT genotypes being observed in patients with GD when compared with control subjects. In the present study, we found no difference between cases and controls in allele or genotype frequencies of the -590 C/T polymorphisms of the IL-4 gene in United Kingdom subjects.
The identification of genetic loci conferring susceptibility to common complex diseases such as GD has proved problematic. The general lack of progress seen with the candidate gene approach has led many researchers to proceed with genome-wide searches in the hunt for susceptibility loci. Chromosomal regions of linkage have been identified in GD and include GD1, GD2, GD3, and chromosome 18q. However, with the exception of the recent positional cloning of the new type 2 diabetes gene encoding calpain-10 (CAPN10) on chromosome 2 (NIDDM1), linkage studies in families in complex diseases have met with limited success, and new candidate genes are awaited. At the present time, therefore, geneticists are using both genome-wide linkage searches and candidate gene studies in attempts to identify new susceptibility loci.
The IL-4 gene does not fall within any region of linkage to GD but was selected previously as a candidate because IL-4 is a T helper cell type 2 cytokine involved in the promotion of humoral immunity. In the original study by Hunt et al. (2), the frequency of the T allele of the C/T polymorphism of the IL-4 gene was 14% in the control subjects and 6% in patients with GD. In the present study, we found the frequency of the T allele in controls to be similar to Hunt et al. (2) at 12%. However, the frequency of the T allele in patients with GD was 14.3%, similar to controls but quite different to that found in patients with GD in the study of Hunt et al. (2).
The differences in the results obtained in the two studies may reflect several factors. There may be genuine geographical differences in the data sets, however, this is unlikely as all patients and controls were white Caucasians from the United Kingdom. This is supported by the fact that the frequencies of alleles and genotypes in the control population are similar in both studies. A more likely explanation for the different findings of the studies relates to the respective size of the data sets. The present study comprises a larger number of both patients with GD (381) and control subjects (285) compared with the study of Hunt et al. (GD = 138, controls = 101; Ref. 2). Using these numbers and an allele frequency of 14% in controls and 6% in patients with GD, Hunt et al. (2) have only 16% power at a significance level of less than 0.001 (as reported in their study) to support their hypothesis that polymorphism of the IL-4 gene is associated with GD. This falls well below the standard level of 80%. In contrast, our data set of 666 individuals has 97% power to detect the same magnitude of difference between cases and controls at a significance level of 0.05. These power calculations suggest the original result to be a random chance event in a small data set and highlight the importance of large data sets when attempting to obtain meaningful results from case control candidate gene association studies.
In conclusion, the present study observed no difference in allele or genotype frequencies of the -590 C/T IL-4 promoter polymorphism between controls and patients with GD, suggesting that this polymorphism does not play a role in the genetic susceptibility to GD in white Caucasians in the United Kingdom.
Acknowledgments
We acknowledge the help of J. Daykin (University of Birmingham), J. Carr-Smith (University of Birmingham), A. Daly (University of Birmingham), M. Armitage (Royal Bournemouth Hospital), A. Hattersley (Royal Devon and Exeter Hospital), and P. Dodson (Birmingham Heartlands Hospital) in recruiting patients for this study.
Footnotes
Address all correspondence and requests for reprints to: Dr. S. C. L. Gough, Division of Medical Science, University of Birmingham, Birmingham Heartlands Hospital, Bordesley Green East, Birmingham B9 5SS, United Kingdom.
This work was supported by the award of a projects grant from the Wellcome Trust (Grants M/95/3717 and 012297), Eli Lilly UK, and the Endowment Fund of the former United Birmingham Hospitals. R.N. is a West Midlands NHS Executive Sheldon Research Fellow.
Abbreviations: GD, Graves’ disease; IL-4, interleukin 4.
Received February 22, 2001.
Accepted April 18, 2001.
References
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  Y. Tomer and T. F. Davies Searching for the Autoimmune Thyroid Disease Susceptibility Genes: From Gene Mapping to Gene Function Endocr. Rev., October 1, 2003; 24(5): 694 - 717. [Abstract] [Full Text] [PDF]
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 B. Vaidya, P. Kendall-Taylor, and S. H. S. Pearce The Genetics of Autoimmune Thyroid Disease J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5385 - 5397. [Full Text] [PDF]
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