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Abstract Record Report 74th Annual Meeting of the American Thyroid Association Millennium Biltmore Hotel, Los Angeles, California October 10 – 13, 2002 Program Number 1 Thyroid Hormone Metabolism The Gene Coding for the Type 3 Iodothyronine Deiodinase Is Imprinted and Required for Normal Neonatal Growth and Survival A. Hernandez1,2, S. Fiering3, E. Martinez1,2, V. Galton2, D. St. Germain1,2 Departments of 1Medicine, 2Physiology, and 3Microbiology and Immunology, Dartmouth Medical School, Lebanon, New Hampshire, USA The mouse Dio3 gene codes for the type 3 deiodinase (D3), a conserved selenocysteine- containing enzyme that inactivates thyroid hormones and is highly expressed in the pregnant uterus, the placenta and the fetus. The Dio3 gene and its human homolog, DIO3, map to chromosomal regions (12F1 and 14q32, respectively) which are known to include imprinted genes. Genomic imprinting refers to monoallelic gene expression and results from epigenetic mechanisms. Using homologous recombination in mouse embryonic stem (ES) cells we have introduced a critical mutation that renders the D3 inactive. No detectable D3 activity was found in the heads and bodies of fetuses (E14 to E18) that were homozygous (-/-Dio3) for the mutation. The D3 activities found in the heads, limbs, liver and bodies of +/-Dio3 fetuses were, respectively, 30%, 21%, 17%, and 22% of those found in +/+Dio3 fetuses when the mutation was inherited from the father. However, when the mutated allele was of maternal origin, D3 activities in the same tissues were essentially the same as those in the wild types, indicating preferential expression from the paternal allele. The weight of -/-Dio3 fetuses is normal during fetal life. However, mild growth retardation was observed in 2-day-old -/-Dio3 neonates (93% of the weight of wild type mice), and it became severe by 14 days of age (65%) and persists into adulthood (80%). Growth retardation was also observed in +/-Dio3 mice that inherited the mutation from the father, but not in those where inheritance came from the mother. Genotyping of the offspring of +/-Dio3 breedings identified 22% of fetuses (173 screened), but only 13% of 2-day-old neonates (205 screened) as -/-Dio3, rather than the expected 25% prevalence. We conclude that the Dio3 gene is preferentially expressed from the paternal allele and required for normal neonatal growth and survival. 74th Annual Meeting of the American Thyroid Association Millennium Biltmore Hotel, Los Angeles, California October 10 – 13, 2002 Program Number 2 Thyroid Diseases NHANES III: Impact of TSH:TPOAb Relationships on Redefining the Serum TSH Normal Reference Range C. Spencer1, J. Hollowell2, J. Nicoloff1, L. Braverman3 1University of Southern California, Los Angeles, California; 2University of Kansas Medical Center, Lawrence, Kansas; and 3Boston University School of Medicine, Boston, Massachusetts, USA BACKGROUND: The conventional serum TSH reference range in euthyroid subjects approximates 0.4 to 4.0 m IU/L. However, several recent reports indicate that this range may be skewed to the high-end by the presence of occult autoimmune thyroid disease. As the NHANES III survey included sensitive TPOAb and TgAb measurements, it affords an opportunity to address this question (Hollowell JCEM 87:489,2002). STUDY DESIGN: 12,974 NHANES subjects [male (M) : female (F) ratio = 1.2:1] with normal total T4 (excluding a history of thyroid disease, thyromegaly, pregnancy, T4 or E2 Rx) were analyzed for TPOAb and TSH relationships. RESULTS: The prevalence of TPOAb for M+F was 11.2% (7.9%M/15.2%F). For males, TPOAb prevalence was lowest (4.9 ± 0.5 se %) in the TSH range 0.4 to 2.0 mIU/L (male reference) and rose progressively from 8.0 ± 1.5%, for the TSH 2.0-2.5 mIU/L interval to 94±4% for TSH>20mIU/L. For females, TPOAb prevalence was lowest (6.1±1.1%) in the TSH range 0.4 to 1.5 mIU/L (female reference) and rose progressively from 13.4±1.8% for the TSH 1.5-2.0 mIU/L interval (p< 0.001) to 98 ± 1% for TSH>20mIU/L. TgAb without TPOAb showed no association with TSH concentration. Serum TPOAb concentrations were increased for all TSH intervals above 2.5 mIU/L for males and above 1.5 mIU/L for females. CONCLUSIONS: (1) Occult thyroid disease is a common occurrence in normal reference populations. (2) Based on TPOAb prevalence, the new normal serum TSH adult reference range approximates 0.4 to 2.0mIU/L. (3) Pilot results of a highly selected normal sub- population (n=76) supports this new reference range (Spencer). (4) This new TSH reference range should be considered as the optimal end-point for T4 replacement Rx. (5) TPOAb measurement is indicated when the TSH value is outside this new range. 74th Annual Meeting of the American Thyroid Association Millennium Biltmore Hotel, Los Angeles, California October 10 – 13, 2002 ID: Program Number 3 Cancer The Follicular Thyroid Carcinoma-associated PAX8/PPAR-γ-1 Fusion Gene Decreases the Rate of Apoptosis and Shortens the Doubling Time of Thyroid Cell Lines J. Powell, X. Wang, I. Hay, Y. Zhao, H. Hiddinga, M. Sahin, N. Eberhardt, B. McIver Division of Endocrinology, Mayo Clinic and Foundation, Rochester, Minnesota, USA A translocation of chromosomes 3p25 and 2q13 is seen commonly in follicular thyroid carcinoma (FTC), resulting in the expression of a fusion protein including the first 9 exons of the thyroid-specific transcription factor PAX-8 with a full length peroxisome proliferator receptor gamma (PPAR-γ). Expression of the fusion protein (designated PPFP) is restricted to FTC, and is seen infrequently in follicular adenoma (FA) and in other thyroid carcinoma histotypes. PPFP is a putative oncogene, but direct evidence for its oncogenic action has not previously been reported. We assessed the impact of PPFP over-expression on an immortalized thyroid cell line (NT cells), following transient transfection. The PPFP gene was engineered into an expression vector (pCDNA3.1), which exhibits constitutive activity in mammalian cells. A normal thyroid cell line, transformed by incorporation of the SV40 large-T antigen (NT cell line), was transfected in triplicate either with PPFP-pCDNA3.1, the papillary thyroid carcinoma oncogene RET/PTC-1 incorporated into the same vector, or the control vector. Viable cell numbers were assessed by Coulter-counter after 12, 24, 48 and 72 hours, and the proportion of cells in early or late apoptosis was determined by flow cytometry after staining with Annexin and 7-AAD. Following transfection with a control vector, NT cells exhibited a doubling time of approximately 66 hours, which was essentially unchanged after transfection with the RET/PTC-1 oncogene. In contrast, after transfection with the PPFP gene, the doubling time was shortened to 41 hours, a decrease of 38% (p<0.001). Similarly, the rate of apoptosis in cells expressing PPFP was reduced by more than 65% at 72 hours, from 18.4% to only 6.3% (p<0.002). The FTC-associated PAX8-PPAR-γ fusion gene reduces apoptosis, enhances cell-survival and shortens the doubling time of thyroid cell lines. These data strongly support a role for PPFP as an important oncogene in follicular thyroid carcinoma. 74th Annual Meeting of the American Thyroid Association Millennium Biltmore Hotel, Los Angeles, California October 10 – 13, 2002 Program Number 4 Thyroid Diseases T4 plus T3 Treatment for Hypothyroidism: A Double-Blind Comparison with Usual T4 A. Levitt, J. Silverberg Sunnybrook & Women's Health Sciences Centre and University of Toronto, Toronto, Ontario, Canada This study compares T4 versus a combination of T4 and T3 treatment in subjects with T4-treated hypothyroidism, with one or more persistent symptom of hypothyroidism. Subjects were euthyroid and had been on stable doses of T4 prior to entry. Subjects received T4 in their current dose or T4 plus T3 (in a physiological ratio of 15:1), in a double blind twice daily dosing over a 3- to 6-month period. Outcome was evaluated once TSH levels were in the normal range for two months, and using two primary outcome measures: the Sunnybrook Hypothyroid Symptom Severity scale (SHSS) and the Clinical Global Impression of improvement (CGI-I). 59 subjects completed the protocol. Using a 2 (group) by 2 (time) ANOVA for repeated measures, there was no main effect of group or a group by time interaction on the outcome measures. However, there was a significant positive main effect of time (SHSS: F = 54.1, p <.0001; CGI: F = 22.2, p <.0001). As expected the combination group had a significantly lower T4 level (t = 4.0, p <.0001) and a significantly higher T3 level (t = 4.1, p <.0001) by the final week as compared with the T4 alone group; however, there were no significant differences in TSH levels. Examination of all subjects showed that those subjects with the greatest reduction in T4 equivalent dosages had the greatest improvement on both outcome measures. Furthermore, improvement in hypothyroid symptoms was correlated with changes in cholesterol (r = .36, p <.05) and LDL-cholesterol (r = .32, p <.05). These data suggest that there is no overall advantage to the combination of T4 and T3 used in this study over T4 alone in hypothyroid patients already on T4 replacement, but that a reduction in overall T4 equivalent dose is associated with improvement in symptoms. 74th Annual Meeting of the American Thyroid Association Millennium Biltmore Hotel, Los Angeles, California October 10 – 13, 2002 Program Number 5 Thyroid Hormone Action The S14 Knockout Mouse Shows Resistance to Diet-induced Obesity C. Mariash1, G. Mucha1, Q. Zhu2, G. Anderson1 1Department of Medicine, University of Minnesota, and 2Eli Lilly Company, Minneapolis, Minnesota, USA We have recently reported that the Spot 14 knockout mouse showed enhanced hepatic lipogenesis when maximally induced by carbohydrate feeding and T3 administration.
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