Vijay Panicker, Ponnusamy Saravanan, Bijay Vaidya, Jonathan Evans, Andrew T. Hattersley, Timothy M. Frayling, and Colin M. Dayan

DOI: http://dx.doi.org/10.1210/jc.2008-1301
Received: June 16, 2008
Accepted: January 26, 2009
Published Online: July 02, 2013

Abstract

Introduction: Animal studies suggest that up to 80% of intracellular T in the brain is derived from circulating T4 by local deiodination. We hypothesized that in patients on T4 common variants in the deiodinase genes might influence baseline psychological well-being and any improvement on combined T4/T3 without necessarily affecting serum thyroid hormone levels.

Methods: We analyzed common variants in the three deiodinase genes vs. baseline psychological morbidity and response to T4/T3 in 552 subjects on T4 from the Weston Area T4[/T3[/ Study (WATTS). Primary outcome was improvement in psychological well-being assessed by the General Health Questionnaire 12 (GHQ-12).

Results: The rarer CC genotype of the rs225014 polymorphism in the deiodinase 2 gene (DIO2) was present in 16% of the study population and was associated with worse baseline GHQ scores in patients on T4(CC vs. TT genotype: 14.1 vs. 12.8, P = 0.03). In addition, this genotype showed greater improvement on T4/T3therapy compared with T4 only by 2.3 GHQ points at 3 months and 1.4 at 12 months (P = 0.03 for repeated measures ANOVA). This polymorphism had no impact on circulating thyroid hormone levels.

Conclusions: Our results require replication but suggest that commonly inherited variation in the DIO2 gene is associated both with impaired baseline psychological well-being on T4and enhanced response to combination T4/T3 therapy, but did not affect serum thyroid hormone levels.

In subjects on thyroid hormone replacement, a D2 deiodinase genotype is associated with poorer psychological well-being and increased likelihood of improvement with addition of triiodothyronine.

Up to 3% of the population in Western countries is on thyroid hormone replacement (1), the majority on T4alone. However, the adequacy of this to replace physiological requirements and reverse patients symptoms remains controversial due to several observations. In thyroidectomized rats, Escobar-Morreale et al. reported that it was not possible to normalize tissue levels of thyroid hormone (T4 and T3) by replacement with T4 alone (2, 3) or T3 alone (4). In humans, patients on T4 monotherapy have a significantly higher serum T4 to T3 ratio for a similar TSH than people with normal thyroid function (5, 6). Some markers of thyroid hormone action, such as IGF-1 may not normalize on T4monotherapy (7). Finally, a significant number of patients report persistent symptoms despite titration of T4replacement to adequate serum levels of thyroid hormone and normalization of TSH levels (8, 9). These observations led to the proposition that combination therapy with both T4 and the T3 might be more effective. However, although in the initial study of combination therapy (T4 and 3, patients treated with the combination appeared to have improved well-being (10), 10 subsequent larger studies failed to confirm this benefit (11, 12, 13, 14, 15, 16, 17, 18, 19, 20) and in at least one case demonstrated a large and sustained placebo effect (18).

Metaanalysis of these trials (21) showed no benefit from combination therapy and a carefully controlled study of overreplacement with T4 also failed to show benefit (22). Doubt remains though because an excess psychological morbidity among patients on T4 has been documented in three separate large community-based studies (9, 23, 24), and also anecdotally there remain patients who feel better on combination therapy.

One possibility that might resolve these issues is the existence of a subgroup of patients who require combination therapy due to an inherited abnormality. If this group represented less than 20% of the population, such patients may be too infrequent for their presence to be detected in the intervention trials but could still account for significant morbidity in patients on T4. The three iodothyronine deiodinases represent possible candidate loci for such genetic variation, because these are responsible for the interconversion of T4 and T3 (25). The Weston Area T4/T3 Study is the largest study of thyroid hormone replacement yet conducted (n = 697) (18). We have taken advantage of the greater statistical power of this study to explore the role of common polymorphisms in the three deiodinase genes in determining psychological well-being and the response to partial T3 replacement.

Subjects and Methods

Sample population

Subjects were 552 people in the Weston Area T4/T3 Study (WATTS) who had DNA available for genotyping (total study participants = 697). The study design has been previously described (18), but briefly, subjects on a stable dose of T4therapy 100 ?g or more per day were recruited from 28 primary care practices in the Weston-superMare and Bristol areas of the United Kingdom and randomized to either combination T4/T3 therapy (original dose minus 50 ?g of T4 and added 10 ?g T3 or original dose of T4 alone. Biochemical, physical, and psychological assessments were made at baseline and 3 and 12 months. The trial was double blinded and results analyzed on an intention-to-treat basis. The study protocol was approved by the local research ethics committee.

Biochemical methods

Serum TSH and free T4(fT4) were measured from a serum sample by RIA (Diagnostic Product Corp., Los Angeles, CA). Free T3 (fT3) was measured by chemiluminescence assay (Elecsys system 1010; Roche Diagnostics, Mannheim, Germany). The laboratory reference ranges were: TSH, 0.34.0 mU/liter; fT4, 1024 pmol/liter; and fT3, 2.87.1 pmol/liter. Coefficients of variation were: TSH, 5.58.0%; fT4, 7.710.0%; and fT3, 11.712.6%.

Tag single-nucleotide polymorphism (SNP) selection and genotyping

We used genotype data from the Caucasian European individuals in the International Haplotype Mapping Project (http://www.hapmap.org) to select a set of SNPs that capture the majority of common variation across the three deiodinase genes (DIO1, DIO2, and DIO3) including 50 kb either side of the genes. We used a minor allele frequency of at least 10%. The 21, seven, and seven SNPs in the DIO1, DIO2, and DIO3 genes required nine, four, and six SNPs, respectively, to capture all common variants with an r2 > 0.8. These were: D1, rs11206237, rs11206244, rs2235544, rs2268181, rs2294511, rs2294512, rs4926616, rs731828, and rs7527713; D2, rs12885300, rs225011, rs225014, and rs225015; and D3, rs1190716, rs17716499, rs7150269, rs8011440, rs945006, and rs1190715. We used only SNPs that were in Hardy Weinberg equilibrium (P > 0.05) and were genotyped in at least 97.5% of the samples in the final analyses. We examined the association between these SNPs and baseline (before randomisation) psychological well-being.

Genotyping was performed by KBiosciences (http://www.kbioscience.co.uk) using their own novel fluorescence-based competitive allele-specific PCR (KASPar). Assays were designed for each of the 19 SNPs by KBiosciences. Design of an assay for the SNP rs1190715 was not possible and two further SNPs (rs1190716 and rs12885300) failed quality control. The percentage of duplicate samples included was 20% and concordance between duplicate samples was 99% or greater. Use of these 16 SNPs resulted in coverage of 100, 85, and 71% of the common (minor allele frequency > 10%), HapMap based, variation in DIO1, DIO2, and DIO3, respectively, at r2 > 0.8.

Psychological assessments

At each visit, the patients psychological well-being was assessed by the following self-report scales: the General Health Questionnaire, 12-question version (GHQ-12) (26, 27), a disease-specific thyroid symptom questionnaire (TSQ) (9), and the Hospital Anxiety and Depression Scale questionnaire (HAD) (28). In addition, patients completed a satisfaction question on a 5-point scale on the second and third visits. The GHQ-12 and TSQ were scored by both linear (Likert method) and categorical methods (score ?3 by GHQ method) (26, 27). HAD is divided into seven questions for anxiety (HAD-A) and seven questions for depression (HAD-D) all scored 03. HAD anxiety or depression caseness was defined as a total score from the seven questions 8 or greater, which has been shown to provide the best sensitivity and specificity for case finding (28).

For response to T4/T3 or T4-only therapy, improvement in GHQ-12 score was used as the primary end point as this was the endpoint that the original study was powered to measure (18). All other measures were analyzed as secondary end points.

Statistical analysis

To ensure there were no significant differences between the two study groups, baseline characteristic means were compared by ANOVA and proportions by ?2 test. Initial analysis of the relationship between psychological well-being and genotype at baseline was performed by linear regression, with total GHQ Likert score as the dependent variable and genotype as the independent variable, with each allele considered additive. Logistic regression was also used with GHQ, HAD-D, or HAD-A caseness as the dependent variable. For response to therapy repeated-measures ANOVA was used with the scales, which were normally distributed (total GHQ Likert score, total TSQ Likert score, and satisfaction question score). The total number of missing values was low (<5%). To ensure this did not create error missing values were imputed using the missing values analysis function on SPSS (Chicago, IL), using regression methods to estimate values and adding a random regression residual. This did not significantly change the original estimates, which are displayed in the results section. For GHQ and TSQ, the scores at 3 and 12 months were the repeated measures, the study treatment arm, and genotype were the between-subject effects and baseline (before randomization) score was adjusted for as a covariate. The repeated-measures analysis includes two-way interactions between study treatment arm and genotype and genotype and baseline score.

For rs225014, genotype was analyzed as both additive (each allele increases the association) and with the T-allele as dominant (TT and TC combined vs. CC) as a dominant effect was suggested by the graphs (Fig. 1) and has been proposed previously (29). For satisfaction score, no baseline score was adjusted for because there was no baseline assessment. No correction was made for multiple testing because, despite being the largest study to date, it is still underpowered to detect all but very large differential gene-treatment effects. Instead, we have chosen to report the P values and associations, which should be considered suggestive, and have qualified our findings by stating clearly that the results need replicating as a risk of type I statistical error exists. Analyses were performed on Stata version 9.0 (www.stata.com) and SPSS version 14.0 (www.spss.com).

Results

Descriptive statistics of the two groups are displayed in Table 1. The treatment groups were not significantly different at baseline in any of the parameters studied.

CONTINUED HERE /…….

Conclusions

Our results require replication but suggest that a commonly inherited variation in the DIO2 gene is associated both with impaired baseline psychological well-being on T4 and enhanced response to combination T4/T3 therapy, but did not affect serum thyroid hormone levels.

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