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Cytotoxic T Lymphocyte-Associated Molecule-4 Gene Polymorphism and Hyperthyroid Graves’ Disease Relapse after Antithyroid Drug Withdrawal: A Follow-Up Study

Division of Endocrinology and Metabolism (P.-W.W., I.-Y.C., R.-T.L., C.-J.H.), Department of Internal Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung 833, Taiwan; and Department of Medical Research (E.H., S.-H.H.J.), Kaohsiung Medical University Hospital, and Graduate Institute of Medical Genetics (E.H., S.-H.H.J.), Kaohsiung Medical University, Kaohsiung 807, Taiwan

Address all correspondence and requests for reprints to: Dr. Suh-Hang H. Juo, Kaohsiung Medical University, 100 TzYou First Road, Kaohsiung City 807, Taiwan. E-mail: shj34{at}columbia.edu.

	Abstract

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Context: We previously showed an association between the exon1 +49 A/G single nucleotide polymorphism (SNP) and the relapse of Graves’ disease (GD). The G allele was associated with early relapse.

Objective: In this follow-up study, we sought to replicate the result by genotyping nine additional polymorphisms and recruiting another 60 GD patients.

Design and Participants: The GD patients were divided into three groups: recurred within 9 months, between 10–36 months, and more than 36 months. There were 65 patients with early recurrence, 55 with medium recurrence, and 88 with late recurrence. Although several SNPs were associated with recurrence, the most significant marker was still exon1 +49 A/G. Separate analysis of the genotypes for the 60 newly enrolled patients indicated that our present study was not biased by the previous samples. Once exon1 +49 A/G was included in the model to predict recurrence, other markers would not add more predictive information. Haplotype analysis did not show an additional value once exon1 +49 A/G was compulsorily included.

Results: Multivariate logistic regression analysis showed that GG genotype of exon1 +49 A/G SNP had an adjusted odds ratio of 2.2 (95% confidence interval, 1.1–4.4) compared with the combined group of GA plus AA. Other significant predictors were large goiter size at the end of the treatment and positive TSH-binding inhibitory Ig at the end of the treatment.

Conclusions: This follow-up study confirms the usefulness of the exon1 +49 A/G SNP of the cytotoxic T lymphocyte-associated molecule-4 gene in predicting recurrence after cessation of treatment. There is no additional power by including other polymorphisms to predict recurrence.

	Introduction

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CYTOTOXIC T LYMPHOCYTE-associated molecule-4 (CTLA-4) provides a negative signal to the T cells and controls the adaptation of T cells to a state of proliferation unresponsiveness and tolerance (1, 2, 3). In addition, CTLA-4 signaling mediates antigen-specific apoptosis of T cells and suppresses autoreactive proliferation of T lymphocytes (4). Many studies conducted over the last decade have lead to consistent results regarding the association of Graves’ disease (GD) with the single nucleotide polymorphism (SNP) at position 49 in exon1 +49 A/G of the CTLA-4 gene (5, 6, 7, 8, 9, 10).

Assuming CTLA-4 expression may influence the progression of an ongoing immune process rather than just disease susceptibility, we previously successfully showed an association between the exon1 +49 A/G SNP and the relapse of GD after drug discontinuation (11). In 148 Chinese GD patients and 171 controls, we found that a significant difference of genotype frequencies (P < 0.001) and allele frequencies (P < 0.001) among the three groups of GD patients with different time of relapse after drug withdrawal. The frequency of the detrimental G/G genotype decreased from 79% to 64% and then 39% in groups of patients with short, medium, and long duration of remission, respectively. The frequency of the G allele was significantly different between controls and patients with early relapse (P < 0.001) but not different between controls and patients with long duration of remission (P = 0.33) (11).

Given the previous intriguing finding, we continued to search for other CTLA-4 polymorphisms that may provide additional power to predict recurrence of GD. We extensively examined another nine publicly available polymorphisms of the CTLA-4 gene (Fig. 1), including three commonly studied polymorphisms: –318C/T (12), the (AT)n in the 3' untranslated region (3'UTR) of exon 4 (9, 13), and CT60 (14, 15, 16, 17). In the present study, we also added another 60 GD patients. With more subjects and genetic markers, we hope to provide more reliable information for GD management in the future.

View larger version (6K): [in this window] [in a new window]   FIG. 1. The locations of 10 examined polymorphisms. SNP rs5742909 is also known as –318 C/T; SNP rs231775 is also known as exon1 +49 A/G; and SNP rs3087243 is also known as CT60.

	Subjects and Methods

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We studied 208 Chinese patients with GD (172 females and 36 males; aged 39 ± 13 yr) recruited from the Endocrine Clinic and 171 healthy controls (78 females and 93 males; aged 53 ± 11 yr) from the Health Screening Center of the Chang Gung Memorial Hospital in Kaohsiung, Taiwan. Among the 208 patients with GD, 148 patients had participated in our previous study and 60 were newly recruited. The diagnosis criteria for GD were: elevated serum T₄ and/or T₃ and suppressed TSH levels; diffusely increased thyroidal uptake of technetium-99m or iodine-131; and the presence of TSH-receptor antibodies and/or antimicrosomal antibodies. Only patients who completed a treatment course of at least 1 yr and had adequate follow-up after drug withdrawal were included. Patients with a history of radioiodine therapy or previous thyroid surgery were excluded. The controls were euthyroid healthy subjects without clinical evidence or family history of any autoimmune disease. The study project was reviewed and approved by the institutional review committee, and informed consent was given by the patients and controls.

Treatment and follow-up

The duration of antithyroid drug treatment extended from 1–3 yr. After drug withdrawal, patients were suggested to follow up at every 3-month interval in the first year and then at every 6-month interval. Relapse was confirmed by the recurrence of symptoms of hyperthyroidism and laboratory data of elevated serum T₄ and/or T₃ exceeding the upper limit of the normal range of our laboratory. Patients with suppressed TSH alone would not be counted as recurrence until they had abnormally elevated T₄/T₃. We used the previous criterion (11) to divide the patients into three groups according to the time of relapse after drug withdraw: group 1, early relapse within 9 months; group 2, relapse between 10–36 months; and group 3, either in remission for more than 3 yr or relapsed after 3 yr of drug withdrawal.

Evaluation of patients

Clinical and laboratory evaluation included the CTLA-4 genotypes; serum levels of T₄, T₃, and TSH; goiter size; and TSH-receptor antibodies at the beginning and end of treatment. We defined goiter size into three grades: grade 1, a palpable goiter not reaching the medial edge of the sternocleidomastoid muscle; grade 2, a palpable goiter reaching the medial edge of the sternocleidomastoid muscle but not exceeding the lateral edge; and grade 3, a palpable goiter exceeding the lateral edge of the sternocleidomastoid muscle. Serum T₄, T₃, and TSH levels were determined by RIA. TSH levels were determined by a one-step sandwich assay with a normal range of 0.254.0 µU/ml (0.254.0 mU/liter) (RIA-gnosthTSH; CIS Bio International, Gif-Sur-Yvette, France) or a chemiluminescent assay with a normal range of 0.54.5 µU/ml (0.54.5 mU/liter) (Nichols Institute Diagnostics, San Juan Capistrano, CA). TSH-receptor antibody was measured as TSH-binding inhibitory Ig (TBII) with a radioreceptor assay (TR-AB; CIS Bio International).

Genotype

DNA was extracted from peripheral blood leukocytes. The SNPs of the CTLA-4 gene were genotyped by the PCR restriction fragment length polymorphism method with proper primers. The 3'UTR microsatellite (AT)n was genotyped using the PCR and a fluorescence-based technique. For each candidate polymorphism, we first screened 40 controls and 40 GD patients to calculate the minor allele frequency before genotyping the overall samples. If the minor allele frequency was 5% or greater, then the overall patients and controls were genotyped. The locations of the examined polymorphisms were illustrated in Fig. 1.

Statistics

Allele frequencies were estimated by direct gene counting. Hardy-Weinberg equilibrium was tested for each polymorphism by the ² test. Comparisons of individual clinical and laboratory variables between groups 1, 2, and 3 were performed with one-way ANOVA for the continuous data, and the ² test or Fisher exact test for the categorical data. Multivariate logistic regression was performed to assess the strength of association between the length of remission and the clinical and laboratory variables. The odds ratio (OR) and its 95% confidence intervals (CI) were calculated by using SPSS version 13.0. A two-tailed P value less than 0.05 was considered statistically significant.

Linkage disequilibrium (LD) between pairs of polymorphisms was estimated by Haploview (18). LD was assessed for any pair of SNPs and haplotype blocks were defined using the default setting of the Haploview software (18). Haplotype analyses were performed using the Hap-Clustering program with adjustment for covariates (19).

	Results

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Among 10 genotyped polymorphisms, four SNPs were monomorphic in the initially screened 40 patients and 40 controls. They were rs16840275 (all G/G), rs3087242 (all G/G), rs231781 (all G/G), and rs13384548 (all G/G). These four SNPs were not genotyped in the remaining samples. Therefore, we focused the results of the remaining six polymorphisms in the current study. The genotypes of the five SNPs were in Hardy-Weinberg equilibrium in the patients and controls. However, the distribution of the genotypes of the (AT)n polymorphism in the 3'UTR was not in Hardy-Weinberg equilibrium (see supplemental Table 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). The distribution of (AT)n was shown in Fig. 2. A total of 20 alleles with different lengths constituted two separate distributions. The first distribution included allele sizes from 80–82 bp and the second included allele sizes from 96–216 bp, whereas allele sizes between 82–96 bp were totally absent in either GD cases or normal controls. The pattern of two distributions was similar between the controls and GD patients. Because of Hardy-Weinberg disequilibrium, we did not present the statistical results from the (AT)n polymorphism.

View larger version (9K): [in this window] [in a new window]   FIG. 2. The pattern of distribution of (AT)n in the 3'UTR.

There were 65 patients in group 1, 55 in group 2, and 88 in group 3 according to the duration of remission. There was a significant difference of genotype distribution (² test, P < 0.001) among the three groups. Group 1 had the highest proportion (78.5%) of the G/G genotype compared with group 2 (60.0%) and group 3 (45.5%). Similarly, there was a clear trend of decreasing frequency of the G allele and increasing frequency of the A allele from group 1 to 3 (P < 0.001) (Table 1). The current result from our 208 GD patients further confirmed our previous observation based on 148 GD patients (11).

The difference of genotype frequency among the three groups of GD patients with different time of relapse existed mainly between group 3 and group 1 (P < 0.001). The difference of genotype frequency was not significant between group 1 and group 2 (P = 0.087) and between group 2 and group 3 (P = 0.10). Figure 3 shows the data obtained by Kaplan-Meier analysis with recurrence being defined as an event. Group 1 GD patients had very a different genotype distribution from controls (P = 0.0003), although the difference between overall GD cases and controls was not significant (Table 1). The results between GD patients and controls were similar to our previous report.

View larger version (10K): [in this window] [in a new window]   FIG. 3. K-M plot shows the proportions of patients who remained in remission. There was significant difference in the rates of recurrence between GG and non-GG groups (P < 0.0003).

For SNP rs5742909 (promoter –318 C/T), there was a significant difference of genotype distribution (P = 0.017) among the three groups of GD patients (Table 1). There was a clear trend of decreasing frequency of the C allele and increasing frequency of the T allele from groups 1 to 3 (P = 0.025). For SNP rs231777, we found only marginal statistical significance (Fisher test, P = 0.065) between the three genotypes and three relapse groups (Table 1). There was a significant trend of decreasing frequency of the C allele and increasing frequency of the T allele from groups 1 to 3 (P < 0.001). We did not genotype this SNP for all controls because the result for three relapse groups was not significant. For SNP rs231779, there was a significant difference of genotype distribution (Fisher test, P = 0.0008) among the three groups of GD patients (Table 1). Because this SNP can be completely tagged by exon1 +49 A/G (r² = 0.99), we did not genotype the entire control samples. For SNP rs3087243 (CT60), the result was a borderline significance (Fisher test, P = 0.039) for the genotype distribution among the three groups of GD patients (Table 1). There was a significant trend of decreasing frequency of the G allele and increasing frequency of the A allele from groups 1 to 3 (P = 0.008). Group 1 GD patients had a different genotype distribution from controls (P = 0.0051), but the difference between overall GD cases and controls was not significant.

Multivariate logistic regression analysis

To be consistent with our previous report (11), we used remission for more than 3 yr to dichotomize remission duration while performing multivariate analysis. The results showed that GG genotype of exon1 +49 A/G SNP had an adjusted OR of 2.2 (95% CI, 1.1–4.4) compared with the combined group of GA plus AA. Other significant predictors in the model included large goiter size at the end of the treatment (grade 2 vs. grade 1: adjusted OR = 2.4; 95% CI, 1.1–5.6; grade 3 vs. grade 1: adjusted OR = 3.0; 95% CI, 1.1–8.2) and positive TBII at the end of the treatment (adjusted OR = 2.9; 95% CI, 1.3–6.6) (Table 2). The GG genotype was the only risk factor that could be assessed before the start of antithyroid drug treatment.

LD and haplotype analysis

SNP rs231775 and rs231779 were in complete LD with r² = 0.99 and D' = 1. The LD for other pairs of SNPs was shown in Fig. 4. SNP exon1 +49 A/G, rs231777 and rs231779 were located in the same haplotype block. Because exon1 +49 A/G SNP is the most significant variant, we performed haplotype analysis for this SNP along with other SNPs. We used haplotype blocks including two or three SNPs for haplotype analyses, and results were less significant than the result from single exon1 +49 A/G SNP. In fact, the results from haplotype analysis were in concert with the regression modeling mentioned above, both of which indicated that exon1 +49 A/G alone is sufficient to explain the CTLA-4 genetic effect.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Pair-wise LD measures of D', its LOD score, and r2 for the SNP. For each cell, the number on the top line is D'; the number on the second line (in parentheses) is the LOD score of D', and the number on the bottom line is r2.

	Discussion

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Exon1 +49 A/G (21) and CT60 SNPs (16) have been shown to have biological relevance. Both of the two SNPs have A and G alleles, and G is the risk allele for either of the two loci. Although Ueda et al. (16) reported that CT60 is the major causal variant in determining mRNA expression, we did not find the susceptible haplotype GG carried a higher risk than single allele G of exon1 +49 A/G polymorphism. This may be due to complex regulation of gene expression (for example other functional locus not considered here or the discrepancy between gene expression and protein levels). According to the SNP database of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/SNP), the allele frequencies of exon1 +49 A/G are very different across ethnicities. The risk G allele of exon1 +49 A/G is more frequent in Asians (65%) than Caucasians (20%) or Africans (29%). Similarly, the risk G allele of CT60 is more common in Asians (70%) and Africans (80%) than Caucasians (50%). Therefore, the two polymorphisms provide different statistical powers for different study populations.

Sahin et al. (22) replicated our previous results in a Turkish population. Similar to our study, they reported that the GG genotype of exon1 +49 A/G SNP had a significant increase of risk for GD recurrence compared with the AA genotype. Their multivariate regression model did not find TBII as a significant predictor for recurrence, although our previous and the present studies showed the predictive value of TBII. Actually, the usefulness of TBII measured at the end of treatment yielded inconsistent results across studies (22, 23, 24). Although serum levels of the posttreatment TSH were also mentioned as a useful predictor for recurrence in some studies (22, 23), our study did not show a predictive value for this parameter. Among all the potential predictors, only CLTA-4 genotypes can be measured before the treatment and thus it can be a superior marker in determining the treatment strategy than other predictors that are measured at the end of treatment.

Another interesting finding that needs to be addressed is the pattern of distribution of the (AT)n polymorphism. The lengths of the observed 20 (AT)n alleles did not comprise one continuous distribution, which causes Hardy-Weinberg disequilibrium. The most common allele in each distribution was 82 and 100 bp, respectively. There is a lack of alleles between 82–96 bp in either GD cases or normal controls, which results in two distinct distributions (Fig. 2). Reviewing the literature regarding the (AT)n polymorphism in GD and other autoimmune diseases, previous publications also showed a same pattern of allele distributions in their study subjects (9, 13, 25, 26, 27, 28). According to the functional assay, the half-life of CTLA-4 mRNA was shorter for the long allele (112 bp) than short allele (86 bp) (28). Although we do not have an explanation for this observation of two discontinuous distributions, the lack of allele between two distributions may imply a survival selection.

In conclusion, this follow-up study confirms the usefulness of the CTLA-4 genotypes in predicting recurrence after cessation of treatment. Patients with the highly recurrent genotype may consider a more aggressive treatment. Although several polymorphisms of CTLA-4 can be used to predict relapse, exon1 +49 A/G is the most informative marker in our population. There is no additional power by including other polymorphisms to predict recurrence.

	Footnotes

 
This work was supported by grants from the National Science Council (NSC 93-2314-B-182A-112).

All the authors (P.-W.W., I.-Y.C., R.-T.L., C.-J.H., E.H., and S.-H.H.J.) have nothing to declare.

First Published Online April 10, 2007

Abbreviations: CI, Confidence interval; CTLA-4, cytotoxic T lymphocyte-associated molecule-4; GD, Graves’ disease; LD, linkage disequilibrium; OR, odds ratio; SNP, single nucleotide polymorphism; TBII, TSH-binding inhibitory Ig; 3'UTR, 3' untranslated region.

Received December 13, 2006.

Accepted March 30, 2007.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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