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Original Studies |
Department of Laboratory Medicine, Kyoto University School of Medicine, Kyoto 606–01, Japan
Address all correspondence and requests for reprints to: Dr. Shinji Kosugi, Department of Laboratory Medicine, Kyoto University School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606–01, Japan. E-mail: kosugi{at}kuhp.kyoto-u.ac.jp
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Abstract |
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C at
nucleotide +1060 in NIS complementary DNA in a male patient who was
born from consanguineous marriage, had a huge goiter, and lacked the
ability to accumulate iodide but was essentially euthyroid. The
mutation results in an amino acid replacement of
Thr354Pro in the middle of the ninth transmembrane
domain. COS-7 cells transfected with the mutant NIS complementary DNA
showed markedly decreased iodide uptake, confirming that this mutation
was the direct cause of the disorder in the patient. Northern analysis
of thyroid ribonucleic acid revealed that NIS messenger ribonucleic
acid level was markedly increased (>100-fold) compared with that in
the normal thyroid, suggesting possible compensation by overexpression. |
Introduction |
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To date, 37 cases from 22 families with this disorder have been reported (1, 2, 3, 4, 5, 6, 7, 8). Diagnosis is made by 1) absence or poor radioactive iodide uptake without iodide overload, 2) defective iodide condensation in salivary and gastric glands, 3) apparent or latent hypothyroidism that becomes evident when iodide intake is restricted and can be restored by iodide administration, and 4) exclusion of other defects in the process of thyroid hormone synthesis. The entity of this disorder is clearly defined, and no diagnostic errors are possible, as Leger et al. pointed out (5).
Recent studies revealed that iodide is totally cotransported with sodium by the Na+/I- symporter (NIS) in the plasma membrane of the thyroid cells (9, 10). NIS plays an initial and rate-limiting step in synthesis of iodide-containing thyroid hormones. NIS concentrates iodide in thyroid cells by active transport countering an electrochemical gradient and maintains iodide concentrations in thyroid cells about 20- to 100-fold those in serum. In 1996, rat (11) and human (12) NIS complementary DNA (cDNA) sequences were reported. The deduced amino acid sequence revealed that NIS is an intrinsic membrane protein with 12 putative transmembrane domains and is a member of Na+/solute cotransporter family. These advances have focused on the NIS gene as a candidate disease gene for iodide transport defect.
Here, we report a loss-of-function mutation of the NIS gene in a patient with iodide transport defect and discuss the pathophysiology and mechanism of compensation by intake of large amounts of iodide.
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Subjects and Methods |
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A male Japanese patient with iodide transport defect was presented in a local report 23 yr ago (3) and was reviewed in Ref. 2 as case 11. He had had no health problems before diffuse goiter was noted at 18 yr of age. At the age of 30 yr, he consulted a hospital complaining of a huge goiter and easy fatiguability, but was diagnosed to be euthyroid. Examination 5 yr later (3) revealed that he had a huge diffuse goiter and was euthyroid, but thyroidal 131I uptake was continuously very low for 72 h after administration; 4% at 3 h and 3% at 24 h. 131I uptake did not increase after administration of 10 U TSH over the successive 3 days. Most of the administered 131I was rapidly excreted in urine. Saliva/serum and gastric juice/serum 131I ratios at 4 h were 1.5 (control, 84.8–130.3) and 0.6 (control, 51.0), respectively. After administration of 125I, open biopsy was performed for in vitro assays. A thyroid specimen was incubated with 131I; the tissue/medium 131I concentration ratio was 0.94 (control, 1.30–1.47), confirming no active transport of iodide in vitro. Analysis of 125I-iodinated amino acids revealed normal organification. Ultracentrifugation of the soluble fraction showed 17S thyroglobulin, suggesting a lack of abnormality in thyroglobulin. Thyroid peroxidase activity was normal.
Present examinations
We reevaluated the patient, who is now 60 yr old. He had no developmental or intellectual problems, was 173 cm in height, and had a body weight of 73 kg. He had a diffuse, elastic-soft, and smooth-surfaced goiter with a transverse diameter of 11.5 cm as detected by ultrasonography. He was clinically euthyroid and lacked eye symptoms and other abnormal findings on physical examination. Laboratory data confirmed that he was euthyroid. No abnormal findings were found in chemical or hematological examinations. Essentially no thyroidal 123I uptake was observed (1.4% at 3 h and 2.5% at 24 h after administration; normal range, 7–35%). Salivary glands showed no accumulation of iodide either. Iodide concentrations in urine and serum were 610 and 64 µg/L, respectively, which are in the high normal ranges for Japanese subjects (13, 14), indicating no overload but relatively high intake of iodide compared with world standard (15). Antibodies against thyroglobulin, thyroid peroxidase, and TSH receptor were negative. We performed open biopsy of the thyroid to examine NIS cDNA with the patient’s informed consent.
Ribonucleic acid (RNA) isolation, reverse transcription-PCR (RT-PCR), and direct sequencing
Total RNA was extracted by the acid guanidinium/phenol/chloroform method from approximately 100 mg thyroid tissue using RNAzol B (Biotecx Laboratories, Houston, TX) from the patient’s and normal thyroid tissues. The normal control thyroid was the tissue surrounding a benign thyroid adenoma removed surgically. RT was performed on 2 µg total RNA (preheated at 70 C for 10 min) in 40 µL reaction buffer [25 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L dithiothreitol, and 5 mmol/L MgCl2] containing 30 pmol oligo(deoxythymidine)15 primer, 20 U avian myeloblastosis virus reverse transcriptase XL (Life Sciences, Petersburg, FL), and 40 U ribonuclease inhibitor (Toyobo, Osaka, Japan) at 42 C for 40 min. For PCR amplification of NIS cDNA (nucleotides -59 to +1975 containing the full-length coding region), 4 µL RT reaction solution were incorporated in 100 µL PCR buffer [60 mmol/L Tris-HCl (pH 8.5), 15 mmol/L (NH4)2SO4, 1.5 mmol/L MgCl2, and 10% dimethylsulfoxide] containing 2 U Taq polymerase (Takara, Tokyo, Japan), 2 U Taq Extender PCR additive (Stratagene, La Jolla, CA), and 50 pmol each of oligonucleotide primers (5'-TTCCCCCGCTTGAGCACGCAGG-3' and 5'-GAGGTTCCATCCCAGGGTGTCAG-3'). Samples were denatured for 5 min at 94 C and then subjected to 30 cycles consisting of 1 min at 94 C, 1 min at 58 C, and 2 min at 72 C. The last extension was carried out for 12 min. Direct full-length sequencing of the purified PCR product was performed using Exo(-)Pfu DNA polymerase (Stratagene) and primers specific to the reported human NIS cDNA sequence (12).
Analysis of genomic DNA
Genomic DNA from the patient, his family members, and normal subjects was extracted from peripheral blood cells using a WB kit (Wako, Tokyo, Japan). A portion of NIS DNA surrounding the identified mutation was amplified by PCR with primers 5'-AAGATCTGCCTGGAGTCC-3' (corresponding to nucleotides +1001 to +1018) and 5'-CAGTGACTGCAGCCATAG-3' (corresponding to nucleotides +1099 to +1082). The purified product that was approximately 1.1 kilobase (kb) in length was subjected to direct sequencing using the same primers.
Construction of expression vectors, transfection, and iodide uptake assay
PCR-amplified mutant and wild-type human NIS cDNAs were directly
inserted by TA cloning into the pCR3.1 vector under control of
cytomegalovirus promoter (Invitrogen, Carlsbad, CA). The full-length
nucleotide sequences of the constructs were confirmed. COS-7 cells were
transfected with 25 µg mutant or wild-type NIS DNA or control vector
DNA (pCR3-CAT, Invitrogen) by electroporation (Bio-Rad, Richmond, CA).
Transfection efficiencies were monitored by cotransfection with pSVGH
and measurement of GH concentration in culture medium. Cells were
aliquoted into 24-well plates (
105 cells/well).
Forty-eight hours after transfection, assays of iodide uptake were
performed as previously described (16, 17). Briefly, cells were
incubated with 125I and 10 µmol/L NaI in HBSS containing
0.5% (wt/vol) BSA and HEPES
(2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)-NaOH at pH
7.4 for 2–5 min at 37 C. After incubation, cells were quickly washed
twice with ice-cold incubation solution without iodide, and subjected
to radioactivity measurement. The assays were repeated three times. The
total cell protein concentration was measured with the Bio-Rad protein
assay system.
Northern analysis
Ten micrograms of total RNA were electrophoresed in 1% agarose gels containing 0.66 mol/L formaldehyde and blotted onto nylon filters (Hybond N+, Amersham, Aylesbury, UK) according to the manufacturer’s instructions. As a mol wt marker, a 0.24- to 9.5-kb RNA ladder (Life Technologies Gaithersburg, MD) was used. Purified full-length NIS cDNA probe (-59 to +1975) and human ß-actin cDNA probe (Nippon Gene, Tokyo, Japan) were radiolabeled by random priming. Hybridization (0.5–1.0 x 106 cpm/mL) and washing were performed as previously described (18); final washings were carried out at 65 C in 1 x SSPE (150 mmol/L NaCl, 10 mmol/L NaH2PO4, and 1 mmol/L ethylenediaminetetraacetic acid, pH 7.4)-0.1% SDS.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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). It was not noted in the original
report (3), but our further interview revealed that his parents were
second degree relatives (cousins; Fig. 1). Histologically, the thyroid specimen
showed diffuse hyperplasia of thyrocytes. All of the above observations
satisfy the criteria for diagnosis of the iodide transport defect
reported by Stanbury et al. (19). To investigate whether the
NIS gene is involved in the iodide transport defect, we examined the
NIS gene in this patient.
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CCA) in the middle of the ninth
transmembrane domain (Fig. 2). No other
nucleotide changes from the reported sequence (12) were found in the
region from nucleotides -37 to +1952 containing the full coding
region. These observations were also confirmed by completely sequencing
12 independent clones obtained by direct TA cloning of the PCR product
in pCR3.1 vector (Invitrogen).
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1.1-kb fragment. Direct sequencing of the PCR product using the same
primers revealed an intron of 1.0 kb between nucleotides +1058 and
+1059 of the NIS cDNA (Fig. 3). The
patient had the same homozygous mutation as that found in cDNA in the
thyroid (Fig. 4). His daughter was
heterozygous for the mutation; she was euthyroid (Table 2) and had no goiter, supporting the
recessive nature of the disease. His wife and 13 normal subjects had no
base substitution in this region (Fig. 4).
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0.4% of that
in cells with wild-type NIS), but still significant,
perchlorate-sensitive iodide uptake compared with that in cells
transfected with control vector (pCR3-CAT, Invitrogen; Fig. 5). Cotransfection with pSVGH and
measurement of GH concentrations in the media showed no differences in
transfection efficiencies.
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). TSH levels at open
biopsy and operation were 6.2 and 3.2 mU/L, respectively. The size of
NIS mRNA in the thyroid was 2.9 kb, similar to that of rat NIS mRNA
in FRTL5 rat thyroid cells (20), and this was not different between the
patient and the normal subject (Fig. 6).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Pro; ACACCA) of the NIS cDNA in a patient with
iodide transport defect who was born from a consanguineous marriage.
This mutation itself was confirmed to be the direct cause of the
disease in this patient by expression experiments that showed markedly
decreased iodide uptake in cells expressing the mutant and by Northern
analysis that showed an increase, not a decrease, in NIS mRNA level in
the patient’s thyroid compared with that in normal thyroid. The amino acid sequence of the ninth transmembrane domain of NIS, in the middle of which the mutation exists, is entirely conserved in rat and human homologs, suggesting a conservative structural and/or functional significance of this domain of NIS. However, we must await detailed three-dimensional characterization by mutagenesis, modeling, and purification before discussing the role of this portion and the kinking effect by proline introduction.
Although the entity of iodide transport defect appears clearly defined as described above, the clinical features of this disorder seem heterogeneous. Some patients show cretinism, and some remain euthyroid without mental or developmental disorders, as in this case (2). Most have goiter, but some cases without goiter have been reported (2). The amount of iodide intake has been suggested to influence the differences in the clinical picture (2). Investigation of the NIS gene in other patients with this disorder might facilitate elucidation of the genotype-phenotype relationship.
The compensation mechanism for poor or lack of iodide accumulation by NIS remains unclear. However, iodide administration was reported to cure hypothyroidism and reduce goiter size (2), as in this case. Transport of iodide through nonspecific channels or carriers might compensate for low uptake if relatively large amounts of iodide are taken (2). Alternatively, in this case very low NIS activity might have been compensated for by overexpression of the gene, although whether the mutant NIS protein is actually overexpressed in the plasma membrane has not been clarified.
Very recently, Kogai et al. (20) reported that TSH increased the level of NIS mRNA in FRTL-5 cultured rat thyroid cells. This increase in message was maximum (up to 8-fold from the basal level without TSH stimulation) at 1 U/L TSH. At the time of open biopsy in our case, the TSH level was slightly increased. However, it cannot account for the marked (>100-fold) increase in the NIS message in the case with iodide transport defect. Our present observations may provide insight into another important mechanism of transcriptional regulation of NIS, such as by iodide itself or forms of organic iodine, as suggested by a mechanism called autoregulation by iodine (21, 22). Further, variability in the clinical picture among cases with iodide transport defect might be explained by the difference in NIS gene expression as well as differences in genotype. It is interesting that Inomata et al. (8) reported siblings born from a consanguineous marriage with different clinical pictures of iodide transport defect. Investigation of NIS gene expression will promote understanding of the pathophysiology not only of the iodide transport defect but also of the iodide deficiency from which tens of millions of people in the world suffer.
The iodide transport defect is usually evident in salivary and gastric glands as well as in the thyroid. Identification of a loss-of-function mutation of the NIS gene in a patient showing no condensation of radioactive iodide in saliva or gastric juice supports the existence of NIS in these tissues, although it has not been proven directly, and the physiological significance of NIS in these tissues is not yet clear.
During the preparation of this manuscript, the identical NIS mutation was reported by Fujiwara et al. in Osaka in a patient with congenital hypothyroidism (23). Our patient has origins from Wakayama prefecture adjacent to Osaka prefecture. Although there was no known familial relationship between these two cases, descendency from a common ancestor origin should be considered. Alternatively, this mutation might be in a hot spot. It is interesting to compare the clinical features between the cases, although these were not documented in detail in their report (23). In their report, cells expressing the T354P mutant showed iodide uptake indistinguishable from that by control cells (23). The discrepancy from the results of our expression study might have been due to low and/or noncontrolled transfection efficiency or lack of comparisons with iodide uptake in the presence of perchlorate. Electrophysiological examinations in the presence of high concentrations of iodide might resolve this discrepancy (24).
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Acknowledgments |
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Footnotes |
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Received July 30, 1997.
Revised August 27, 1997.
Accepted September 9, 1997.
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