Pathological findings in dyshormonogenetic goiter with defective lodide transport

Clinical Research

Pathological Findings in Dyshormonogenetic Goiter with Defective Iodide Transport Rosalinda Y. A. Camargo, MD, Jorge Luiz Gross, MD, Sandra P. Silveiro, MD, Meyer Knobel, MD, and Geraldo Medeiros-Neto, MD Abstract An adult male patient (38 yr old) with a large congenital goiter with hypothyroidism was suspected of having a defective iodide (I-) transport mechanism based on low thyroid uptake and a very low salivary/plasma ratio. Moreover the high serum levels of TSH (104 I~U/mL) declined to 7.2 I~U/mLwith a corresponding normalization of serum total T4 concentration, after 40 d of treatment with Lugol's solution (6 mg I/d). Thyroid surgery was performed because a fine-needle aspiration biopsy of a nodule revealed atypical cells associated with the presence of a large compressive goiter (150 g). Pathologic examination indicated histological findings compatible with the hyperplastic pattern with predominant microfollicular aspect. Immunostaining for other specific thyroid proteins, thyroid peroxidase (TPO) and Tg, indicated normal expression of both transcripts. Electron microscopy (x13,000) showed the ultrastructural aspects of a hyperactive follicular cell with folding of the basal membrane. Sequencing of the entire sodium/iodide (Nail) symporter (NIS) cDNA derived from thyroidal mRNA revealed a homozygous substitution of the normal cytosine in nucleotide 1163 with an adenine, resulting in a stop signal at codon 272. This nonsense mutation produces a truncated NIS symporter protein without iodide transport activity. Although the propositus is homozygotic for the NIS-272X expression of one normal allele in the heterozygotic son, mother, and paternal aunt is sufficient to maintain normal thyroid function. Key Words: NIS gene; congenital goiter; hypothyroidism; iodide transport; genetic mutations.

Thyroid Unit, Division of Endocrinology, University of S~o Paulo Medical School (RYAC, JLG, MK, GM-N); Division of Endocrinology, Hospital Clinicas de Porto Alegre (SPS), Universidade Federal do Rio Grande do Sul, Brazil. Address correspondence to Dr. Geraldo Medeiros-Neto, Thyroid Unit, Endocrinology, Hospital das Clinicas, C Postal 3061, 01060-970, S~o Paulo, Brazil. E-mail: consular@ embratel.net.br

Endocrine Pathology, vol. 9, no. 3, 225-233, Fall 1998 9 Copyright 1998 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/98/9:225-233/$10.25

Introduction A system for neonatal screening for congenital hypothyroidism in most Western countries has shown an incidence of this syndrome in approximately 1 in 4000 newborns [1]. In the majority of cases, hypothyroidism is caused by thyroid agenesis or dysgenesis (either ectopic thyroid tissue or hypoplastic thyroid gland). Molecular studies in these patients have failed to detect mutations in the gene for thyroid transcription factor 1 [2,3] but have indicated the possibility that muta-

tions in the thyroid stimulating hormone (TSH) receptor gene are involved [4]. In approximately 15-20% of the cases the hypothyroid state results from defects of thyroid hormonogenesis inherited in an autosomal recessive manner. Such defects have been traced to abnormalities in all known steps involved in thyroid hormone synthesis [5]. Active iodide (I) transport, the first step in the biosynthesis of thyroid hormone, is mediated through the sodium/iodide symporter that received the acronym of

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Fig. 1. Correlation of the exon-intron structure of human sodium/iodide symporter gene with a map of the 12 transmembrane domains. The exons in the coding region are represented by hatched boxes, the 5' and 3'untranslated regions are represented by open boxes. Exons are linked to the corresponding transmembrane domains (TM) of the hNIS protein by dotted lines (from ref. 8).

NIS. Located in the basolateral membrane of the thyrocyte, this protein, with a predicted molecular mass of=65 kDa, belongs to the family of sodium-dependent cotransporters, has a structure of presumably 12 transmembrane segments (Fig. 1), with both amino and carboxy termini inside the cell [6]. The human NIS gene has been mapped to chromosome 19 and consists of 15 exons encoding a protein of 643 amino acids [7,8]. Since the first report in 1958 of defective iodide transport in a hypothyroid child [9], about 42 additional cases, belonging to 29 families and occurring world-wide, have been reported [reviewed in ref. 5]. Determination of the molecular mechanism of this form of hypothyroidism had to await the cloning of the rat NIS gene [6] followed by the cloning of the human NIS cDNA [7,8]. Very recently, four mutations of the NIS gene have been reported producing a NIS protein with no detectable function [10-13]. We reported here a congenitally hypothyroid patient with an iodide-transport trapping defect that was found to be homozygous for a nonsense mutation in the NIS gene [13]. Biochemical and pathological

studies have confirmed the defective iodide transport system, whereas other thyroid proteins (thyroid peroxidase [TPO], TSH receptor, and thyroglobulin [Tg]) were presumably normal.

Material and Methods Propositus

A 39-yr-old man presented with a large goiter. He was born to healthy Caucasian parents, from Portuguese ancestry that denied consanguinity. Thyroid enlargement was noted at 3 mo of age with mild clinical symptoms of hypothyroidism. Treatment with increasingdoses of L-thyroxine (L-T4) was given over the years. Physical and mental development proceeded normally and the patient completed his high school education. Nevertheless, the goiter continuously increased in size. On physical examination at 23 yr of age, the thyroid gland was estimated to weigh about 150 g (10-fold the normal size). A nodule, 8 cm in diameter, was found in the right lobe of the thyroid gland and another, 2 cm in diameter and very hard in consistency, was found in the left lobe.

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Laboratory results while the propositus was not taking the prescribed amount of>T4, revealed a low serum T4 of 3.7 gg/dL (normal range: 5.0-10.5 lag/dL) with a high thyrotropin (TSH) level of 104 ~U/mL, compatible with hypothyroidism. Thyroid peroxidase and thyroglobulin antibodies were negative, indicating the lack of autoimmune thyroid disease. Low 24 h thyroidal radioiodide uptake of radioactive iodine of 1% (normal range: 18-32% at 24 h) and a low saliva to plasma (S/P) radioiodide ratio of 3 (normal > 28) 60 min after the intravenous (iv) administration of a radioiodide tracer led to the diagnosis of Itrapping defect. The latter test showed that the inability of the thyroid gland to concentrate I- was also present in the salivary gland that normally shares this NIS-dependent function with the thyroid. Treatment with iodine only (6 mg/d) was begun. Forty days later the s e r u m T 4 concentration has increased to 6.3 Jag/dL and the TSH declined to 7.2 ~U/mL. The dose of iodide was doubled, resulting in a further increase of the serum T4 level to 8.9 blg/dL, a decrease inTSH to 0.5 gU/mL, and a small reduction of the thyroid gland size. A fine-needle aspiration of the two thyroid nodules revealed atypical cells with considerable nuclear pleomorphism similar to that present in thyroid malignancy. For this reason, the patient's gland was removed surgically, tt weighed 158 g, and on microscopic examination showed small follicles with scant colloid that were lined by tall columnar cells, suggesting intense stimulation of the gland. There was no lymphocytic infiltration. Following thyroid surgery, euthyroidism is being maintained with 150 big L-T4/d. The patient's mother, a paternal aunt, and his son were clinically euthyroid and had normal or slightly enlarged (less than 20 g) thyroid glands. All gave informed consent to undergo studies according to

resolution 196/96 from the National Health Council, Ministry of Health, Brazil. Serum tests of thyroid function, including total and free T4, total tri-iodothyronine, and TSH were in the normal range for all relatives. Tests Performed In Vivo: SIP (Saliva/Plasma) Ratio

SIP ratio was measured by a modification of the method of Harden et al. [14]. Fifteen and 60 min after the iv administration of 5 laCi of Na131I, blood was sampled from the opposite arm simultaneously with the collection of saliva. The S/P ratio of radioiodide was determined by counting equal volumes of these fluids.

Thyroid Tissue Thyroid tissue removed at surgery from the propositus was divided in four fragments. One was for histological examination and fixed in buffered formalin. A second fragment was separated for electron microscopy and fixed in osmic acid. A third (and larger) portion of the thyroid gland was immediately frozen and kept at-85~ for molecular studies. A fourth fragment was sent to the lab for tissue culture and studies on iodide uptake in vitro. Genomic DNA Genomic DNA was extracted from lymphocytes from 30 mL of blood drawn from the propositus and his relatives, using the Ficol Paque plus (Pharmacia Biotech, Piscataway, NJ). Thyroid Iodine Uptake In Vitro The thyroid tissue specimen was brought at once in ice to the laboratory and was cut as quickly as possible with the Stadie microtome. As a control tissue, we have used a presumably normal thyroid tissue surrounding a "cold" nodule that was removed during the same morning and similarly

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prepared. Approximately 150 mg of thyroid tissue was added to culture flasks containing 4 mL of modified Krebs-Ringer phosphate buffer and approx 0.1 btCi (microCurie) of 13IIwithout added carrier. After incubation at 37~ for 1 h, the slices were removed, rinsed in cold saline, weighed, homogenated in 2 mL of buffer, and taken for determination of labeled iodine in a well scintillating counter. Aliquots of the suspending medium were similarly counted and expressed as counts/min/mg of tissue. The ratio between the counts/rain for mg of tissue to counts/min/mg of medium was established (T/M ratio). TPOActivity and To Separation by Gel Filtration TPO activity was measured in the microsomal fraction of the thyroid tissue by the tri-iodide method as previously described [15]. The particulate fraction was suspended in 1 mL digitonin (1% w/v) and incubated at 4~ for 48 h. The dig/ton/ntreated suspension was centrifuged and the supernatant, containing solubilized TPO, was used for the assay. An aliquot of the supernatant (• g) was submitted to Sepharose C 16 B gel illtration column as previously described [15]. Pathological Examination and Immunohistochemical Studies The morphologic study was carried out using H&E stain. The specimens were fixed in buffered formalin for 20, and can be as high as 70. In nearly half of the affected patients, the S/P ratios were found to be near 1.0. In other half, it ranged from 1.5-5.9, suggesting a partial defect. Another critically important diagnostic piece of information derivesfrom the response to iodine therapy. Although patients with defective iodide transport respond to L-T4 replacement therapy, they should respond equally well to iodine alone. This has been successful in most of the cases [16] and, as seen in the present case, the euthyroid state was rapidly restored. Goiter size, however, may not reduce with iodine administration, as observed in the present report. This may be owing to persistent relativelyelevated levels of serum TSH (around 3.0 mUlL) in spite of normal serum concentrations of free T4 and total T4. Other thyroid proteins involved in the thyroid hormone synthesis and release are normally expressed and functionally active. The TPO activity in the removed thyroid tissue was about three times the normal level and clearly present in histochemical studies. Also a normal eluted peak of

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poorly iodinated thyroglobulin was obtained by gel filtration. It can be concluded from these studies that only the iodine transport system is affected by a genetic defect in this patient. Tissue slices cut at random from the thyroid tissue specimen partially failed to accumulate radioactive iodine from the medium in which they were incubated. However, some radioactivity was present in the homogenized tissue in relation to the medium and this was interpreted as simple diffusion of the tracer into the incubated tissue. In similar experiments conducted by others [16], the T/M ratio ranged from 0.99-1.9. It is also possible that some radioactivity remained in the homogenized tissue without actually being inside of the follicular cell. The pathological aspect of the iodide transport defect has been reviewed byWolff [16] and Medeiros-Neto and Stanbury [5]. The general picture of the histological pattern in surgical specimens obtained from these patients has been one of many, usually small, nodules or adenomas with frequent polymorphism from one structure to another. There is usually evidence of extensive stimulation of the parenchyma, cells tend to be columnar, follicles small, colloid scant or absent, and nuclei may be atypical. The marked nuclear abnormalities often lead to the suspicion of malignancy. In all cases of defective iodide transport examined, there were adenomas of the microfollicular pattern. That suggested characteristics of fetal/embryonal adenoma. The reason for scant or absent colloid is obscure because these glands are usually under elevated TSH stimulation. Moreover, a normal thyroglobulin peak after gel filtration was observed in the soluble protein fraction of the thyroid tissue of this patient, confirming that Tg is normally expressed although poorly iodinated.

The molecular defect in this patient was a mutation in the Sodium Iodine Symporter protein gene, as previously reported by us [13]. Sequencing of the entire NIS cDNA derived from thyroidal mRNA revealed a homozygous substitution of the normal cytosine in nudeotide 1163 with an adenine, resulting in a stop (TGA) at codon 272. This nonsense mutation produces a truncated NIS with undetectable iodide transport activity when expressed into COS-7 cells. This severely truncated mutant protein is not likely to be expressed in the cell basal membrane. The mutation creates a endonuclease (HgA1) recognition site. Digestion of the allele containing the mutant 1163A produces two fragments, 133 and 131 bp (see Fig. 4). Genotyping confirmed that the propositus was homozygous for the mutation, whereas his unaffected mother, son, and paternal aunt were heterozygous. Although the homozygous mutant causes goiter and hypothyroidism, expression of one normal allele in the heterozygote is sufficient to maintain active thyroidal iodine uptake and function. In conclusion we present a patient with defective iodine transport, that was also confirmed by in vivo tests of low S/P ratios and relatively low T/M ratios. Other thyroid proteins (TPO and Tg) were normally expressed and functionally active. The molecular basis for the defect was a truncated NIS protein owing to a premature stop codon at position 272X.

Acknowledgments We gratefully acknowledge the molecular studies conducted by Joachin Pohlenz and Samuel Refetoff (Univ. of Chicago, Chicago, IL). This work was supported by a Research Grant, Project 96/00998-4 from FAPESP (S~_o Paulo, Brazil). The labora-

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Fig. 4. Pedigree and genotype of the propositus and members of his family. The mutation creates a new restriction site for endonuclease HgA I A264 bp fragment was amplified from genomic DNA and digested with this enzyme. The mutant allele produces 2 fragments, 133 and 131 bp. Note that the propositus is homozygous for this mutation showing both fragments but not the 264 bp fragment. By contrast, his mother, a paternal aunt, and his son carry both the wild-type and the mutant allele and have normal iodide-transport mechanism (adapted from ref. 13).

tory work of Maria Silvia Cardia and the expert secretarial work of Maria Suzette Pott are gratefully acknowledged.

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