Identification of a mutation in the coding sequence of the human thyroid peroxidase gene causing congenital goiter.

Thyroid peroxidase (TPO) is the key enzyme in the synthesis of thyroid hormones, and the TPO defects are believed to be the most prevalent causes of the inborn errors of thyroid metabolism. We investigated an adopted boy with iodide organification defect, who presented with florid hypothyroidism at the age of 4 mo, poorly complied with thyroxine treatment, and developed a compressive goiter necessitating partial resection at the age of 12 yr. Biochemical studies revealed the absence of TPO activity in the resected tissue. Genomic DNA studies identified a 4 base-pair insertion in the eighth exon of the TPO gene, and showed that the patient was homozygous for this frameshift mutation. The direct genetic diagnosis of this mutation can be made by digestion of polymerase chain reaction products with NaeI restriction enzyme. This will help assessing its prevalence among the heterogenous genetic group of TPO defects.

Studies on the goiter inhibiting action of iodolactones.

The thyroid gland synthesizes 6-delta-iodolactone, a compound shown to inhibit goiter growth in vivo and cell proliferation in culture. The present studies were performed to characterize this effect further with the aim of exploring the possible therapeutic action of iodolactones. Prevention assay: rats were treated simultaneously with a goitrogen, methylmercaptoimidazole, and either 6-delta-iodo-lactone or 14-iodo-omega-lactone, a synthetic derivative, given either i.p. or p. o. Both compounds caused a significant decrease in thyroid weight irrespective of the route of administration, but oral administration was less effective. A dose-response relationship was observed, the minimal effective dose (i.p.) being 3 micrograms/day. Involution assay: goiter was first induced with methylmercaptoimidazole and then the iodolactones were injected. Both compounds caused a significant involution, which was dose-related. Acute (10 days) administration of the iodolactones did not produce significant changes in several serum parameters (total T3 and T4, cholesterol, total protein, urea and acetylcholinesterase). These results give further support to the potential therapeutic application of iodolactones.

Thyroid autoregulation: evidence for an action of iodoarachidonates and iodide at the cell membrane level.

Iodolipids are the possible mediators of excess iodide in thyroid autoregulation. Previous work from our laboratory has shown that 14-iodo-15-hydroxy-5,8,11 eicosatrienoic acid (I-HO-A) and its omega lactone (IL-w) mimic the inhibitory action of excess iodide upon several parameters of thyroid metabolism. The present experiments were performed in order to study the mechanism of the inhibitory effect of I-HO-A and IL-w on 2-deoxy-D-glucose (DOG) and aminoisobutyric acid (AIB) uptake by calf slices. I-HO-A, IL-w and KI 0.1 mM caused a 33, 31 and 25% inhibition, respectively, of AIB uptake. The presence of 0.1 mM methimazole (MMI) only reversed the effect of KI. The transport of DOG was inhibited by both compounds: I-HO-A caused a 62% decrease, while IL-w produced a 64% inhibition; and MMI failed to relieve their action. On the contrary, the 33% inhibition caused by KI disappeared when MMI was present. Taking into account that AIB and DOG transport across the membrane requires energy, supplied by Na-K-ATPase, changes in its activity were studied. TSH (10 mU/ml) produced a 74% increase in the enzyme activity which was significantly blocked by KI (82%), I-HO-A (100%) and IL-w (100%). Basal enzyme activity was impaired by IL-w (33%), but not by KI. These results were correlated with the decrease of DOG uptake produced by 1 mM ouabain. Tissue specificity effect of iodoarachidonates was demonstrated by the absence of action on DOG transport in kidney and liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism.

Thyroid autoregulation has been linked to an organified iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined. The following pure synthetic compounds were prepared: 1) 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-OH-A); 2) its omega lactone (IL-omega); 3) 5-hydroxy-6-iodo-8,11,14-eicosatrienoic acid delta lactone (IL-delta). Their action on iodine metabolism was studied. Iodine uptake was measured in calf thyroid slices. At 10(-4)M I-OH-A caused a 64% decrease in the T/M ratio, while IL-omega inhibited it by 36% and IL-delta was without effect. At 10(-5)M the inhibition was 44% for I-OH-A and 19% for IL-omega, while T3 was without action. A possible isotopic dilution effect was excluded, and no change in iodine efflux was observed. The inhibition by I-OH-A of iodide uptake was observed after only 15 min preincubation. This compound also decreased 125I accumulation in rats. In calf thyroid slices, I-OH-A at 10(-4)M, inhibited PB125I formation by 80%, IL-omega by 62% and IL-delta by 37%. T3 and arachidonic acid were without action. I-OH-A also caused a dose-dependent inhibition of TSH-stimulated iodide organification. The present results demonstrate, for the first time, that iodinated derivatives of arachidonic acid inhibit thyroid function and mimic the effect of iodide on thyroid autoregulation.

Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.

Thyroid autoregulation. Inhibition of goiter growth and of cyclic AMP formation in rat thyroid by iodinated derivatives of arachidonic acid.

Thyroid autoregulation has been related to intraglandular content of an unknown putative iodocompund. Data from different laboratories have shown that the thyroid is capable of producing different iodolipids, including iodinated derivatives of arachidonic acid; such as 5-hydroxy-6-iodo-8, 11, 14-eicosatrienoic-delta-lactone (IL-delta). Previous results from our laboratory showed that a semi-purified preparation of iodinated arachidonic acid exerts an inhibitory action in vitro on calf thyroid. In the present studies three purified iodinated derivatives of arachidonic acid were synthesized: IL-delta; 14-iodo-15-hydroxy-5, 8, 11-eicosatrienoic acid (I-OH-A) and its corresponding omega-lactone (IL-omega). Their action on MMI-induced goiter was studied in rats. Administration of MMI to rats during 10 days increased thyroid weight by 124%. This effect was significantly inhibited by the simultaneous injection of 5 micrograms/day of I-OH-A (57% inhibition of MMI action), IL-W (39%), IL-delta (33%) and T3 (95%), while arachidonic acid was without action. No inhibition was found with 1.25 micrograms/day Kl, a dose equivalent to that which could be originated from total dehalogenation of the iodocompounds. These results support the idea that these iodocompounds have an intrinsic biologic activity and that there is a correlation between action and chemical structure. Serum TSH was increased around 15-20 fold after MMI administration. Chronic or acute injection of I-OH-A failed to alter TSH levels, indicating that this iodocompound exerts its action directly on the gland, without altering TSH concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on the antigoitrogenic action of iodoarachidonates.

Previous studies have shown that iodoarachidonates (IAs) prevent goiter production in rats. In the present studies we show that both IL-d and IL-w (IAs bearing the iodine atom at the positions 6 and 14, respectively), cause a significant involution of preformed goiter. This effect was evident when IAs were administered either orally or via i.p., although the first one required larger doses to obtain the same degree of inhibition. No changes were observed in serum protein, urea, cholesterol, cholinesterase, T3 or T4. In vitro studies with FRTL-5 cells showed that both IAs inhibit iodide and alpha-AIB uptake, as well as ATPase activity.

Differential levels of thyroid peroxidase and thyroglobulin messenger ribonucleic acids in congenital goiter with defective thyroglobulin synthesis.

The biosynthesis of thyroid hormones requires iodide, thyroid peroxidase (TPO), thyroglobulin (Tg) and H2O2. We have studied two sisters with congenital large goiters and hypothyroidism. Perchlorate tests were negative. Serum T3 and T4 were decreased, TSH was increased and Tg was within the lower limit of normal. Biochemical and molecular studies were performed on goiter samples obtained after surgery. Tg content in both tissues was negligible. Paper chromatography of labeled iodocompounds showed a decrease in T4, and the presence of a pronase/pancreatin-resistant iodoprotein. TPO activity was normal in the tissues. Sephacryl S-300 gel filtration demonstrated labeled iodoalbumin-like protein and the absence of a Tg peak. Salting out studies of soluble protein fraction gave an abnormal pattern. Agarose gel electrophoresis showed the presence of an iodoalbumin-like protein and the absence of Tg in the tissues. This last finding was confirmed by immunoelectrophoresis. The Tg and TPO mRNAs levels were also analyzed. Dot-blot hybridization studies with pM5 (TPO cDNA) and phTgM2 (Tg cDNA) probes showed increased and decreased signals, respectively. The increase in TPO mRNA can be explained as a compensatory mechanism vis a vis an increase in serum TSH caused by decreased serum T3 and T4 due to the impairment in Tg mRNA. The Tg mRNA of both patients was further studied with four different probes covering 5' and 3' regions (phTgM1, phTgB1, phTgB2 and phTgB3). Hybridization was observed with all four probes, thus excluding a dramatic deletion defect. Northern transfer showed a clear signal of hybridization with the phTgB1 probe in the 8-9 Kb range. We may conclude that the biochemical and molecular abnormality of these patients is characterized by a decrease of Tg mRNA and of Tg translation.