Type 2 Deiodinase Polymorphism (Threonine 92 Alanine) Predicts l-Thyroxine Dose to Achieve Target Thyrotropin Levels in Thyroidectomized Patients

Abstract

Type 2 deiodinase (D2), by catalyzing the intracellular conversion of l-T₄ to T₃, plays a major role in thyroid hormone metabolism (1). D2 generates T₃ in several human tissues, including brown fat, brain, pituitary (thyrotroph cells), thyroid, and aortic smooth and skeletal muscle. T₃ concentration at both hypothalamic and pituitary level is the major determinant of the inhibitory control of TSH secretion; therefore, D2 activity plays a pivotal role in the negative feedback regulation of pituitary TSH secretion by circulating T₄ (2–6). A recent hypothesis, based on anatomical distribution, suggests that T₄ is taken up in hypothalamic glial cells and transformed, by D2 activity, into biologically active T₃, which subsequently bounds to TRH producing neurons in the paraventricular nucleus (7). Indeed, compared with normal animals, serum TSH levels are two times higher in D2 knockout mice (1, 8). The D2 gene is located on chromosome 14q24.3 (3). A common missense variant of the gene, in which a threonine (Thr) becomes an alanine (Ala) at codon 92 (D2 Thr92Ala), has been identified and associated with decreased D2 enzyme velocity (4, 9). The few available studies have not evidenced correlations between this polymorphism and either plasma thyroid hormones and TSH levels, or well-being and neurocognitive function in treated hypothyroid patients (5, 10–12). Given the central role of D2 in the feedback regulation of TSH secretion and the reduced enzymatic activity described for the Ala92 variant, this study was set up to investigate whether the D2 Thr92Ala polymorphism predicts the T₄ dosage needed to obtain target TSH levels in thyroidectomized patients.

Patients and Methods

A total of 217 consecutive patients, attending the thyroid clinics of our institutions, were enrolled. Inclusion criteria were the following: age 70 yr or younger, absence of thyroid tissue, absence of systemic or gastroenteric illnesses, and no medications known to interfere with gastrointestinal T₄ absorption. To have homogenous circulating thyroid hormone levels, which help to analyze hypothalamic/pituitary feed back response to T₄ administration, individuals with increased free T₃ (fT₃) and/or free T₄ (fT₄) serum levels were not included in the study. All patients underwent, at least 12 months before entering the study, near-total thyroidectomy, followed by radioiodine residual tissue ablation given for treating differentiated thyroid cancer. When entering the study, all patients were considered disease free (undetectable serum thyroglobulin, negative neck ultrasonography, absence of distant metastases). Immediately after radioiodine treatment, all patients started T₄ therapy (Eutirox, Bracco, Italy) taken under fasting conditions, aimed at obtaining, according to an individual cost to benefit ratio as indicated by the European Thyroid Cancer Taskforce (13), either suppressed (supp) (n = 117, < 0.1 mU/liter) or near-supp (n = 74, ≥ 0.1 < 0.5 mU/liter) serum TSH levels in the presence of normal serum fT₃ and fT₄ levels. To avoid the potential influence of food on T₄ absorption, subjects were recommended to wait at least 1 h before eating and drinking, except water. The study population comprised 191 patients (33 males and 158 females; age 50.2 ± 11.9 yr, range 16–70). All patients were on stable T₄ dose treatment for at least 4 months. The mean T₄ dose in the whole group was 1.9 ± 0.4 μg/kg. The study was approved by the Ethical Committees of our institutions, and all subjects provided written informed consent.

Serum determination of fT₃, fT₄, and TSH was obtained in all patients and reverse T₃ in 107 of 191 subjects. Fasting blood samples were collected at 0800–0900 h before taking T₄ tablets. Serum was separated by centrifugation and stored at −40 C. All determinations were performed simultaneously in the same laboratory. fT₃, fT₄, and TSH were measured by electrochemiluminescence (Elecsys; Roche Diagnostics GmbH, Mannheim, Germany). fT₃ had a normal range of 2.8–7.1 pmol/liter (1.8–4.6 pg/ml), analytical sensitivity of 0.4 pmol/liter (0.26 pg/ml), and intraassay and interassay variations of 4.8 and 6.3%, respectively. fT₄ had a normal range of 12–22 pmol/liter (0.9–1.7 ng/dl), analytical sensitivity of 0.30 pmol/liter (0.023 ng/dl), and intraassay and interassay variations of 1.7 and 3.3%, respectively. TSH had a normal range of 0.2–4 mU/liter, functional sensitivity of 0.014 mU/liter, analytical sensitivity of 0.005 mU/liter, and intraassay and interassay variations of 2.1 and 3.3%, respectively. Reverse T₃ was assessed by RIA (Biocode-Hycel, Liège, Belgium). The normal range was 0.14–0.54 nmol/liter (0.09–0.35 ng/ml), analytical sensitivity 0.01 nmol/liter (0.009 ng/ml), and intraassay and interassay variations 8.5 and 6.2%, respectively. Genomic DNA was extracted from peripheral blood according to standard procedures. The Thr92Ala D2 polymorphism was screened with the ABI PRISM SNaPshot Multiplex Kit (4323151; Applied Biosystems, Foster City, CA) based on the dideoxy single-base extension of an unlabeled primer. The PCR product (255 bp) was amplified using the sense primer (5′-CTCAGGGCTGGCAAAGTCAAG) and antisense primer (5′-CCACACTCTATTAGAGCCAATTG), and purified by digestion with Exo I and SAP enzymes. The primer extension reaction was performed with fluorescently labeled dideoxy-nucleotides and the following primer (5′-TACCATTGCCACTGTTGTCACCTCCTTCTG). The primer-extension reaction product was electrophoresed on the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) after purification by digestion with CIP enzyme. Data were analyzed with GeneScan Analysis Software version 3.1 and GeneScan-120 LIZ size standard analysis parameter (Applied Biosystems). Genotypes of frequent (minor allele frequency ≥ 10%) single nucleotide polymorphisms covering the entire D2 gene were obtained from the International HapMap Project (www.hapmap.org).

Deviation from Hardy-Weinberg equilibrium was analyzed using the χ² test. Data were analyzed using SPSS 12.0 for Windows (SPSS, Inc., Chicago, IL) and are presented as mean ± sd. Differences between groups were calculated using the χ² test or ANOVA with Bonferroni test for post hoc analysis, as appropriate. Multivariate regression analysis was performed to exclude the effect of possible confounding factors, such as age and gender. A statistical difference less than 0.05 was considered significant.

Results

Characteristics of the patients according to genotype are summarized in Table 1. Allelic frequency of Thr92Ala in the study population was 0.39, similar to that previously described in Caucasians (4), and was in Hardy-Weinberg equilibrium. To achieve individual target TSH level, patients homozygous for the allele Ala (Ala/Ala) needed a higher T₄ dose (μg/kg) compared with Ala/Thr and Thr/Thr patients, either considered separately (2.08 ± 0.43 vs. 1.89 ± 0.32 and vs. 1.92 ± 0.39 μg/kg, respectively; P < 0.05 for both) or, according to a recessive genetic model, considered as a single group named X/Thr (2.08 ± 0.43 vs. 1.90 ± 0.35 μg/kg; P < 0.05) (Table 1 and Fig. 1). No difference in serum fT₃, fT₄, and reverse T₃ levels (the latter measured in 19 Ala/Ala and 88 X/Thr) was found between Ala/Ala and X/Thr individuals. The difference between the two groups in T₄ dose needed to achieve target TSH was observable in the near-supp group (P = 0.002), but not in the supp group (P = 0.4), and it remained unchanged after correction for possible confounding factors, including age and gender (Fig. 1).

Fig. 1.

Differences in T₄ doses between two genotypes (Ala/Ala and X/Thr). X/Thr = Thr/Thr + Ala/Thr. The near-supp group (n = 74) contained patients with serum TSH levels at least 0.1 but less than 0.5 mU/liter; the supp group (n = 117) contained patients with serum TSH levels less than 0.1 mU/liter. *, P < 0.05. **, P = 0.002. N.S., Not significant.

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Discussion

Due to the important role of D2 in T₃ production and, consequently, in the feedback regulation of TSH secretion, we hypothesized that the Thr92Ala D2 polymorphism predicts the T₄ dosage needed to obtain target TSH levels in thyroidectomized patients. As a matter of fact, our results indicate that an approximate 20% higher dose is needed in Ala/Ala homozygous subjects for target TSH levels to be reached, thus suggesting a reduced pituitary feedback due to abnormal D2 hypothalamic/pituitary activity. This association was detectable in patients on near-suppressive therapy, but not in those on completely suppressive therapy. Although caution should be used because near-suppressive TSH levels are not the same in all patients (i.e. range 0.1–0.5 mU/liter), thus making this subgroup not perfectly homogeneous in terms of TSH levels, these data suggest that the reduced D2 activity of Ala/Ala individuals can be compensated by higher T₄ administration. Of note, although Ala/Ala subjects received a higher mean T₄ dose, they only exhibited a slightly, not significantly, higher fT₄ serum level compared with the X/Thr group; this is likely due to the limited number of Ala/Ala patients and/or the different distribution of “suppressed” and “near suppressed” subjects in the two groups.

The Ala92 variant has been recently reported to have reduced deiodinase activity in both human thyroid and skeletal muscle tissues (9). In contrast, no significant differences in D2 activity between the Thr92 and Ala92 variants were observed in functional analysis performed in transfected HEK-293 and COS cells (9, 10). Whether the discrepancy observed between data obtained in human tissues or cultured human embryonic or monkey kidney cells is secondary to species and/or tissue specificity needs to be investigated better.

No association has been reported between Thr92Ala polymorphism and either circulating thyroid hormone levels (5, 11), or related clinical features, including self-reported well-being and neurocognitive function (12). There are several differences between these studies and our present report. First, in all these studies, thyroid hormone levels were, by definition, within the wide normal range, thus making it difficult to detect differences in their concentrations across different genotype groups. Second, circulating T₃ levels depend only partly on D2 activity, being heavily modulated by other factors, including thyroid gland production (which, very likely, is partly preserved also in autoimmune hypothyroid patients as were individuals studied in Ref. 12) and clearance rate. Therefore, it is somehow expected that a modest difference in deiodinase activity across the different D2 genotype groups is not easy to be detected by simply measuring thyroid hormone levels in patients with normal or even residual thyroid tissue. To overcome these problems that have probably affected these previous studies, our present investigation was focused on the feedback response of pituitary TSH to suppressive or near-suppressive T₄ doses, in the absence of thyroid tissue; in this setting, the local (hypothalamic and pituitary) D2-dependent deiodination of T₄ to T₃, rather than circulating T₃, has to be considered the major determinant of the inhibitory control of TSH secretion (2–5). Finally, an association of Thr92Ala polymorphism, higher serum TSH levels, and hypertension susceptibility in euthyroid adults was recently reported, and these data seem to support the evidence of the critical role of pituitary D2 in the feedback regulation of TSH secretion (14). The results we obtained refer to T₄ dose per kg body weight. It has been recently proposed that T₄ replacement correlates better with lean than total body mass (15). Therefore, further studies are needed to investigate whether the role of D2 polymorphism in predicting T₄ dose is also observable when lean rather than total body mass is considered. In conclusion, our data clearly suggest a role of the D2 Thr92Ala polymorphism in predicting the need of higher T₄ intake in thyroidectomized patients. If this finding is confirmed in additional, possibly prospective studies, it may predict the T₄ requirement to suppress TSH on the basis of the individual genetic background.

Acknowledgments

We thank Davide Mangiacotti for his skillful assistance.

This work was supported by the Fondazione Umberto Di Mario ONLUS. Cosimo Durante is a Ph.D. Fellow in the Endocrinology and Molecular Medicine Program at the University of Rome “La Sapienza.”

Disclosure Statement: The authors have nothing to disclose.

Abbreviations

 
• Ala,

  Alanine;

 
• D2,

  type 2 deiodinase;

 
• fT₃, free T₃; fT₄, free T₄; supp,

  suppressed;

 
• Thr,

  threonine.

References

Author notes