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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0451
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 11 6296-6299
Copyright © 2005 by The Endocrine Society

BRIEF REPORT

Polymorphisms in Type 2 Deiodinase Are Not Associated with Well-Being, Neurocognitive Functioning, and Preference for Combined Thyroxine/3,5,3'-Triiodothyronine Therapy

Bente C. Appelhof, Robin P. Peeters, Wilmar M. Wiersinga, Theo J. Visser, Ellie M. Wekking, Jochanan Huyser, Aart H. Schene, Jan G. P. Tijssen, Witte J. G. Hoogendijk and Eric Fliers

Departments of Endocrinology and Metabolism (B.C.A., W.M.W., E.F.), Psychiatry (E.M.W., J.H., A.H.S.), and Cardiology (J.G.P.T.), Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands; Department of Internal Medicine (R.P.P., T.J.V.), Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands; and Department of Psychiatry (W.J.G.H.), VU University Medical Center, 1007 MB Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Wilmar M. Wiersinga, M.D., Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, F5-171, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: w.m.wiersinga{at}amc.uva.nl.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Introduction: Some patients on levothyroxine replacement display significant impairment in psychological well-being, compared with sex- and age-matched controls. Levothyroxine-treated patients can be assumed to derive T3 exclusively from deiodination of T4, which, in the central nervous system, is regulated by type II deiodinase (DII).

Objective: We investigated whether two recently identified polymorphisms in the DII gene (DII-ORFa-Gly3Asp and DII-Thr92Ala) are determinants of well-being and neurocognitive functioning and associated with a preference for replacement with a combination of T3 and T4.

Methods: Genotypes for both polymorphisms were determined in 141 patients with primary autoimmune hypothyroidism, adequately treated with levothyroxine monotherapy and participating in a randomized clinical trial comparing T4 therapy with T4/T3 combination therapy. Questionnaires on well-being and neurocognitive tests were performed at baseline.

Results: Allele frequencies in patients with primary hypothyroidism were similar to those of healthy blood bank donors (32.0 vs. 33.9% for DII-ORFa-Gly3Asp and 40.4 vs. 38.8% for DII-Thr92Ala). DII polymorphisms were not associated with measures of well-being, neurocognitive functioning, or preference for combined T4/T3 therapy.

Conclusion: The DII-ORFa-Gly3Asp and DII-Thr92Ala polymorphisms do not explain differences in well-being, neurocognitive functioning, or appreciation of T4/T3 combination therapy in patients treated for hypothyroidism.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
ALTHOUGH MOST PATIENTS with hypothyroidism are satisfied with levothyroxine treatment, some patients remain with complaints. Indeed, a community-based study confirmed that patients, even with a normal TSH, display significant impairment in psychological well-being, compared with sex- and age-matched controls (1). It cannot be excluded that this reflects the effects of having a chronic disease, but alternatively it may indicate that the current mode of treatment with levothyroxine is not the most appropriate one for some patients.

In 1999 a clinical trial reported that combined therapy with T4 and T3 resulted in improved mood, well-being, and cognitive functioning, compared with T4 alone, and that the combination was preferred by most patients (2). Several subsequent studies did not confirm these results (3, 4, 5, 6). In contrast, two recent clinical trials (7, 8) demonstrated subjective preference for combined T4/T3 therapy over T4 monotherapy, but this outcome in favor of T4/T3 therapy was not reflected in any of the secondary outcome measures, which included questionnaires on well-being and a substantial set of neurocognitive tests. In our own trial, preference of patients was correlated with weight loss, which was significant in patients on combined T4/T3 treatment; indeed, 44% of patients who preferred the combination had serum TSH less than 0.11 µg/ml (8).

In patients on levothyroxine therapy, T3 is derived almost exclusively from T4 because the secretion of T3 by the thyroid is probably very limited. In the brain, conversion of T4 to T3 is regulated by type II deiodinase (DII). Recently two polymorphisms in the DII gene have been identified: DII-Thr92Ala (9) and DII-ORFa-Gly3Asp (10). Subtle changes in enzyme activity linked to these DII polymorphisms may have important consequences for T3 availability in the brain. Studies on subclinical hypothyroidism show that subtle changes in thyroid hormone bioavailability may have clear effects on well-being and neurocognitive functioning (11, 12). The aim of the present study was therefore to investigate whether DII polymorphisms are a determinant of well-being and neurocognitive functioning in hypothyroid patients on levothyroxine and whether these genotypes are associated with a preference for replacement therapy with a combination of T3 and T4 in patients participating in our recently published trial (8).


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Blood was collected from 141 patients (18–70 yr of age) with autoimmune primary hypothyroidism on T4 replacement for at least 6 months, with a serum TSH between 0.11 and 4.0 mU/liter as determined in morning blood samples before levothyroxine intake. The patients participated in a randomized, controlled trial investigating superiority of therapy with a T4/T3 combination in a weight ratio of 5:1 or 10:1 over therapy with T4 alone (8). The protocol was approved by the institutional review board of our academic medical center; all patients provided written informed consent.

Well-being of patients was measured by means of self-report questionnaires and neurocognitive functioning by tests administered by a trained psychometrician under supervision of a clinical neuropsychologist, all performed before the study medication of the randomized trial was supplied (8).

Subjective appreciation of the study medication was rated after 15 wk, compared with usual T4 medication from before the trial. A dichotomy was made between those who preferred study medication over their usual medication (i.e. somewhat or much better) and those who did not. Study medication was preferred to usual treatment by 29.2, 41.3, and 52.2% in the T4 alone, T4/T3 10:1 ratio, and T4/T3 5:1 ratio groups, respectively ({chi}2 for trend, P = 0.024) (8).

Blood samples were collected in the morning in the fasting state before taking T4 tablets. Serum TSH and free T4 (fT4) were measured by time-resolved fluoroimmunoassay and serum T4 and T3 by in-house RIA methods. DNA was extracted from 200 µl blood using the MagNaPureLC DNA isolation kit (Roche Diagnostics, Almere, The Netherlands). DNA concentration was measured at 260 nm by GeneQuant (Pharmacia Biotech, Uppsala, Sweden); all samples were diluted to 40 ng/µl (stock) and 5 ng/µl (work solution). Purity was determined by measuring the 260:280 nm ratio. Allelic discrimination was performed to determine genotypes in 5 ng genomic DNA, using the Taqman allelic discrimination assay (Applied Biosystems, Nieuwerkerk aan den IJssel, The Netherlands). Primer and probe sequences were optimized by using the SNP assay-by-design service of Applied Biosystems (for details, see http://store.appliedbiosystems.com). Reactions were performed with the Taqman Prism 7900HT 384-well format.

Data were analyzed using SPSS 11.5 for Windows (SPSS, Inc., Chicago, IL). Deviation from Hardy-Weinberg equilibrium was analyzed using a {chi}2 test. Differences between groups were compared by means of {chi}2 and ANOVA as appropriate. Statistical significance was defined as a two-tailed P < 0.05.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Allele frequencies of DII Thr92Ala and ORFa variants in our T4-treated hypothyroid patients were 40.4 and 31.9%, respectively. Distribution of genotypes did not deviate from the Hardy-Weinberg equilibrium. No association was found between thyroid hormones and either polymorphism (Table 1Go).


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  		TABLE 1. DII genotype frequencies and association with thyroid hormone parameters

 
Patients homozygous for the Thr92Ala polymorphism had the worst scores on all but one of the well-being questionnaires, but the difference between genotypes was significant for only one of the 21 subscales tested (Profile of Mood States anger subscale). Concerning the ORFa polymorphism, in general the homozygotes had the most favorable well-being questionnaire scores, but the mean scores were not statistically different between the groups (Table 2Go).


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  		TABLE 2. DII genotypes and scores on well-being questionnaires (POMS, MFI, Rand, SCL-90) and neurocognitive tests (cognitive speed, attention, memory)

 
With regard to neurocognition, homozygotes for Thr92Ala again had generally the worst scores. However, the difference between groups was significant for only one of the 21 subtests. Among the ORFa genotypes groups, there was no group consistently scoring better or worse. On only one of the subtest scores, the difference between groups was significant (Table 2Go).

To examine whether the polymorphisms were associated with a preference for combined therapy with T3 and T4 over conventional therapy with T4 alone, an analysis was performed among the 92 patients who received a combination of T4 and T3 for 15 wk. Proportion of patients preferring combined T4/T3 treatment was not significantly associated with Thr92Ala (41, 53, 39% in wild type, heterozygotes, and homozygotes) or ORFa (46, 49, 43% in wild type, heterozygotes, and homozygotes) genotypes. This did not change when the analysis was performed separately for the 5:1 and 10:1 ratio groups.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
This study is the first to test the hypothesis that DII polymorphisms are linked to dissatisfaction with levothyroxine treatment in hypothyroid patients or with preference for combined therapy with T4 and T3 over T4 alone. Because the brain is dependent on DII for its local T3 production out of T4, DII activity may be a key determinant of well-being and neurocognitive function. To date two DII polymorphisms, Thr92Ala and ORFa-Gly3Asp, have been described. The Thr92Ala polymorphism is a highly frequent A/G polymorphism at nucleotide 674 of the D2 sequence predicting a change in amino acid 92 of the protein (9, 13). The second polymorphism is in the most upstream short open reading frame (ORFa-Gly3Asp) of the 5' untranslated region of DII (10). The allele frequencies of both polymorphisms found in the present study were quite similar to those reported previously in 158 healthy blood donors: the allele frequency of the Thr92Ala variant in blood donors was 38.8% (9), in our population 40.4%, and that of ORFa-Gly3Asp 33.9 (10) and 31.9%, respectively. Therefore, these polymorphisms are unlikely to be related to the development of primary hypothyroidism.

Our study revealed no association between DII polymorphisms and well-being as measured by means of several questionnaires. Neither were DII polymorphisms related to subjective preference for combined T4/T3 treatment above usual T4 treatment.

It is presently unclear whether the two DII polymorphisms have functional consequences for DII enzyme activity. Previous studies have shown that Thr92Ala polymorphism is related to insulin resistance (13, 14), whereas the ORFa-Gly3Asp polymorphism was associated with lower serum T4 and fT4 but unaltered TSH and T3 levels (10). Based on these data, it has been suggested that Thr92Ala might be associated with lower and ORFa-Gly3Asp with higher DII enzyme activity (10, 13, 14). The lack of association between DII polymorphisms and serum T4 and fT4 in the present study is likely explained by treatment with T4 in our patients, in whom the T4 dose is the prime determinant of serum (free) T4 concentrations.

In summary, the DII-ORFa-Gly3Asp and DII-Thr92Ala polymorphisms do not explain differences in well-being, neurocognitive functioning, or preference for T4/T3 combination therapy among patients with treated hypothyroidism. Thus, the determinants of dissatisfaction with conventional T4 treatment and a preference for T4/T3 combination therapy remain to be elucidated.


   Footnotes
 
First Published Online September 6, 2005

Abbreviations: DII, Type II deiodinase; fT4, free T4.

Received March 2, 2005.

Accepted August 26, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Saravanan P, Chau WF, Roberts N, Vedhara K, Greenwood R, Dayan CM 2002 Psychological well-being in patients on ‘adequate’ doses of l-thyroxine: results of a large, controlled community-based questionnaire study. Clin Endocrinol (Oxf) 57:577–585[CrossRef][Medline]
  2. Bunevicius R, Kazanavicius G, Zalinkevicius R, Prange Jr AJ1999 Effects of thyroxine as compared with thyroxine plus triiodothyronine in patients with hypothyroidism. N Engl J Med 340:424–429
  3. Clyde PW, Harari AE, Getka EJ, Shakir KM 2003 Combined levothyroxine plus liothyronine compared with levothyroxine alone in primary hypothyroidism: a randomized controlled trial. JAMA 290:2952–2958[Abstract/Free Full Text]
  4. Sawka AM, Gerstein HC, Marriott MJ, MacQueen GM, Joffe RT 2003 Does a combination regimen of thyroxine (T4) and 3,5,3'-triiodothyronine improve depressive symptoms better than T4 alone in patients with hypothyroidism? Results of a double-blind, randomized, controlled trial. J Clin Endocrinol Metab 88:4551–4555[Abstract/Free Full Text]
  5. Siegmund W, Spieker K, Weike AI, Giessmann T, Modess C, Dabers T, Kirsch G, Sanger E, Engel G, Hamm AO, Nauck M, Meng W 2004 Replacement therapy with levothyroxine plus triiodothyronine (bioavailable molar ratio 14:1) is not superior to thyroxine alone to improve well-being and cognitive performance in hypothyroidism. Clin Endocrinol (Oxf) 60:750–757[CrossRef][Medline]
  6. Walsh JP, Shiels L, Lim EM, Bhagat CI, Ward LC, Stuckey BG, Dhaliwal SS, Chew GT, Bhagat MC, Cussons AJ 2003 Combined thyroxine/liothyronine treatment does not improve well-being, quality of life, or cognitive function compared to thyroxine alone: a randomized controlled trial in patients with primary hypothyroidism. J Clin Endocrinol Metab 88:4543–4550[Abstract/Free Full Text]
  7. Escobar-Morreale HF, Botella-Carretero JI, Gomes-Bueno M, Galan JM, Barrios V, Sancho J 2005 Thyroid hormone replacement therapy in primary hypothyroidism: a randomized trial comparing L-thyroxine plus liothyronine with L-thyroxine alone. Ann Intern Med 142:412–424[Abstract/Free Full Text]
  8. Appelhof BC, Fliers E, Wekking EM, Schene AH, Huyser J, Tijssen JG, Endert E, van Weert HC, Wiersinga WM 2005 Combined therapy with levothyroxine and liothyronine in two ratios compared with levothyroxine monotherapy in primary hypothyroidism: a double blind randomized controlled clinical trial. J Clin Endocrinol Metab 90:2666–2674[Abstract/Free Full Text]
  9. Peeters RP, van Toor H, Klootwijk W, de Rijke YB, Kuiper GG, Uitterlinden AG, Visser TJ 2003 Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and iodothyronine levels in healthy subjects. J Clin Endocrinol Metab 88:2880–2888[Abstract/Free Full Text]
  10. Peeters RP, van den Beld AW, Attalki H, Toor H, de Rijke YB, Kuiper GG, Lamberts SW, Janssen JA, Uitterlinden AG, Visser TJ 2005 A new polymorphism in the type 2 deiodinase (D2) gene is associated with circulating thyroid hormone parameters. Am J Physiol Endocrinol Metab 289:E75–E81
  11. Cooper DS 2001 Clinical practice. Subclinical hypothyroidism. N Engl J Med 345:260–265[Free Full Text]
  12. Toft AD 2001 Clinical practice. Subclinical hyperthyroidism. N Engl J Med 345:512–516[Free Full Text]
  13. Mentuccia D, Proietti-Pannunzi L, Tanner K, Bacci V, Pollin TI, Poehlman ET, Shuldiner AR, Celi FS 2002 Association between a novel variant of the human type 2 deiodinase gene Thr92Ala and insulin resistance: evidence of interaction with the Trp64Arg variant of the ß3-adrenergic receptor. Diabetes 51:880–883[Abstract/Free Full Text]
  14. Canani LH, Capp C, Dora JM, Meyer EL, Wagner MS, Harney JW, Larsen PR, Gross JL, Bianco AC, Maia AL 2005 The type 2 deiodinase A/G (Thr92Ala) polymorphism is associated with decreased enzyme velocity and increased insulin resistance in patients with type 2 diabetes mellitus. J Clin Endocrinol Metab 90:3472–3478[Abstract/Free Full Text]



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