LONG-TERM FOLLOW-UP OF A PATIENT WITH SPORADIC NONAUTOIMMUNE HYPERTHYROIDISM DUE TO A THYROTROPIN-RECEPTOR MUTATION (D619G)

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1 Case Report LONG-TERM FOLLOW-UP OF A PATIENT WITH SPORADIC NONAUTOIMMUNE HYPERTHYROIDISM DUE TO A THYROTROPIN-RECEPTOR MUTATION (D619G) Eijun Nishihara, MD 1 ; Mamiko Tsugawa, MD 2 ; Yoshikazu Ozaki, MD 2 ; Yuji Nagayama, MD 3 ; Shuji Fukata, MD 1 ; Mitsuyoshi Hirokawa, MD 1 ; Mitsuru Ito, MD 1 ; Mitsushige Nishikawa, MD 1 ; Hirotoshi Nakamura, MD 1 ; Yoshiya Ito, MD 4 ; Akira Miyauchi, MD 1 ABSTRACT Submitted for publication April 13, 2017 Accepted for publication June 20, 2017 From the 1 Center for Excellence in Thyroid Care, Kuma Hospital, Kobe, Japan, 2 Ikeda Municipal Hospital, Osaka, Japan, 3 Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan, and 4 Faculty of Nursing, Japanese Red Cross Hokkaido College of Nursing, Kitami, Japan. Address correspondence to Dr. Eijun Nishihara, Kuma Hospital, Shimoyamate-dori, Chuo-ku, Kobe , Japan. nishihara@kuma-h.or.jp. DOI: /EP CR To purchase reprints of this article, please visit: Copyright 2018 AACE. Objective: Sporadic nonautoimmune hyperthyroidism caused by germline mutations of the thyrotropin receptor (TSHR) gene is a very rare disease that manifests as severe congenital hyperthyroidism. We describe a case presenting with sporadic nonautoimmune hyperthyroidism during adolescence. Methods: Laboratory data, imaging, histopathology, clinical follow-up, and DNA sequencing were evaluated. Results: The patient was born at term with a normal birth weight but showed increased growth velocity at approximately 7 months. He had no medical consultation before tachycardia was detected at a school physical examination at the age of 13 years. Thyroid function tests confirmed hyperthyroidism with negative anti-tshr antibodies. After maintaining methimazole therapy for 12 years, he underwent total thyroidectomy, and the weight of the resected thyroid tissue was 131 g. The subsequent histopathologic findings showed an encapsulated follicular adenoma, adenomatous goiter, and hyperplastic changes of the follicular epithelium without stromal lymphoid infiltration in normal parenchyma. Sequence analysis of the TSHR gene showed two compound heterozygous germline variants (R310H and D619G) on different alleles. The TSHR- R310H was a nonfunctioning variant from the maternal allele. The TSHR-D619G was previously detected as a somatic mutation in patients with toxic adenomas. The TSHR-D619G mutation, which was absent in his parents, was a de novo mutation on the paternal allele. Conclusion: A TSHR germline mutation (D619G) was identified as the cause of sporadic nonautoimmune hyperthyroidism in a Japanese patient. This patient showed no obvious complications until the age of 13 years, but longterm follow-up with methimazole treatment showed further goiter and benign nodular formation. (AACE Clinical Case Rep. 2018;4:e84-e89) Abbreviations: MMI = methimazole; TRAb = thyrotropin-receptor antibody; TSHR = thyrotropin receptor INTRODUCTION Nonautoimmune hyperthyroidism is a very rare disease, which is caused by constitutive activation of germline mutations of the thyrotropin receptor (TSHR) gene. This disease is subdivided into two categories: familial nonautoimmune hyperthyroidism because of autosomal dominant transmission, and sporadic nonautoimmune hyperthyroidism because of de novo mutations in the TSHR gene. To date, 18 cases of sporadic nonautoimmune hyperthyroidism have been described with 14 different mutations in the TSHR gene (1), (see TSH Receptor Mutation Database II, tsh/; SSFA-GPHR, Compared to familial cases, the clinical features of sporadic cases show an earlier onset and an increased severity of e84 AACE CLINICAL CASE REPORTS Vol 4 No. 1 January/February 2018 Copyright 2018 AACE

2 Copyright 2018 AACE Nonautoimmune Hyperthyroidism, AACE Clinical Case Rep. 2018;4(No. 1) e85 hyperthyroidism. Indeed, half of the sporadic cases were diagnosed as overt hyperthyroidism at the neonatal period, and the other half were diagnosed within 2 years after birth because of clinical presentation, and treatment was immediately initiated (1-3). In addition, delayed and insufficient therapy leads to irreversible complications such as craniosynostosis and mental retardation (4). The severity of hyperthyroidism in sporadic cases may be attributed to higher levels of constitutive activity of TSHR than that in familial cases (5) and has prevented familial transmission. In this report, we describe a hyperthyroid patient with a constitutively active de novo TSHR germline mutation (D619G) who started antithyroid drug treatment at the age of 13 years. CASE REPORT The patient was the first child of healthy unrelated parents born at 39 weeks of gestation. His weight, length, and head circumference were 2,860 g, 53 cm, and 32.5 cm, respectively. While his height presented with over +2 SD from 7 months, his weight and head circumference were approximately +1 SD throughout 3 years (Fig. 1). Thereafter, there was no medical consultation before tachycardia was detected at a school physical examination at the age of 13 years. Further examination showed tachycardia was caused by hyperthyroidism, and methimazole (MMI) therapy was subsequently started. Once euthyroidism was achieved using MMI therapy, hyperthyroidism re-occurred whenever an attempt was made to reduce the therapy dosage, which had been maintained for 12 years (Fig. 2). Anti-TSHR antibodies (TRAb) assessed by second-generation TRAb (normal: <10%) and third-generation TRAb (normal: <1.9 IU/L) assays and a bioassay (thyroid-stimulating antibody [TSAb]) (normal: <180%) were negative throughout the clinical course. Goiter had gradually developed, and thyroid nodular formation was apparent at the age of 17 years (Fig. 2). The patient s intelligence was normal, and he graduated from high school. At the age of 25 years, he consulted our hospital while receiving MMI (17.5 mg/day). The patient was 176 cm tall and weighed 61 kg and showed no ocular symptoms. His thyroid function tests showed that serum thyrotropin was µiu/ml (normal, 0.3 to 5.0 µiu/ml), free thyroxine was 0.57 ng/dl (normal, 0.70 to 1.60 ng/dl), and free triiodothyronine was 3.13 pg/ml (normal, 1.7 to 3.7 pg/ml). Anti thyroid peroxidase antibodies, antithyroglobulin antibodies, and anti-tshr antibodies were negative in a third-generation TRAb assay (normal, <1.9 IU/L) and TSAb (normal, <120%). Ultrasonography of the neck revealed an enlarged thyroid gland with multiple nodules that were a maximum diameter of 4.4 cm (Fig. 3 A). After temporarily stopping MMI treatment, the radioiodine uptake at 24 hours was 52.8%, and scintiscan imaging showed radioiodine uptake was relatively faint in the nodular lesions of both lobes (Fig. 3 B). The patient underwent total thyroidectomy, and the resected thyroid tissue weighed 131 g (Fig. 3 C). The subsequent histopathologic findings of the thyroid specimens revealed that the nodular lesions were an encapsulated follicular adenoma (Fig. 3 D) and adenomatous goiter. The nonnodular parenchyma was composed of various-sized thyroid follicles with follicular swelling and papillary projection but without lymphoid infiltration in the stroma (Fig. 3 D and E). His parents were euthyroid, and no goiter was shown on ultrasonography of the neck. DNA Sequencing The present study was approved by the ethics committee of Kuma Hospital, and informed consent was obtained from the patient and his parents for the use of samples for research purposes. Direct sequencing of the TSHR gene and subcloning of PCR products were performed as previously described (6) and according to the manufacturer s protocol using a TOPO TA cloning kit (Invitrogen), respectively. Sequence analysis of genomic DNA of the patient showed two heterozygous single-base substitution at codons 310 and 619 of TSHR (CGC CAC, R310H; GAT GGT, D619G) (Fig. 4). The TSHR-R310H mutation was present as homozygous in his mother but absent in his father. The TSHR-D619G mutation was absent in his parents. The TSHR-R310H mutation was deemed benign or neutral by PolyPhen2 ( pph2/index.shtml), PROVEAN ( seq_submit.php), and PANTHER ( org/tools/csnpscoreform.jsp). In addition, ClinVar ( demonstrated that the allele frequency of a missense variant (929 G>A; R310H) was To clarify the allelic localization of TSHR-R310H and TSHR-D619G in this patient, we subcloned PCR fragments spanning the entire exon 10, and 10 individual clones were sequenced. The results showed that this patient is a compound heterozygote, indicating that individual clones contained either TSHR-R310H or TSHR-D619G (data not shown). Therefore, the de novo TSHR-D619G mutation was on the paternal allele. DISCUSSION We report the case of a patient with sporadic nonautoimmune hyperthyroidism with two compound heterozygous germline variants (R310H and D619G) in the TSHR gene. Both variants have been identified for the first time in nonautoimmune hyperthyroidism. In fact, TSHR- D619G was previously described as a somatic mutation in toxic adenomas (7,8). Compared with the wild type, TSHR-D619G showed an approximately 2.5-fold increase in basal cyclic adenosine monophosphate (camp) levels. However, there was no constitutive activation of the inosi-

3 e86 Nonautoimmune Hyperthyroidism, AACE Clinical Case Rep. 2018;4(No. 1) Copyright 2018 AACE Fig. 1. Growth charts for height, weight, and head circumference from birth to the age of 3 years. tol phosphate cascade (7,9). Although functional characterization of TSHR-R310H is still lacking, it was regarded as a nonfunctioning variant by several protein databases, which is compatible with euthyroidism in the patient s mother carrying the TSHR-R310H homozygous variant. In addition, a de novo TSHR-D619G mutation occurred on the paternal allele independently from the TSHR-R310H mutation on the maternal allele. These findings confirmed the constitutive activation of TSHR-D619G as a direct cause of hyperthyroidism in the present case. The most common clinical findings of hyperthyroidism in the prenatal, neonatal, or early childhood periods are prematurity, low birth weight, marked bone age acceleration, craniosynostosis, and mental retardation (4). Although an increase in growth velocity at approximately 7 months in this patient may suggest a phenotype of congenital hyperthyroidism, he was born at term with a normal birth weight and showed no severe phenotypes such as craniosynostosis or mental retardation before treatment. These findings suggest that clinical features of congenital hyperthyroidism in this patient, if any, were very mild. Indeed, the TSHR-D619G mutation showed a relatively small increase in constitutive activity among the TSHR mutations in sporadic nonautoimmune hyperthyroidism (5). Comparison of the constitutive activity index (basal camp/receptor number) showed that TSHR-D619G activity was almost equivalent with TSHR-D617Y in the third intracellular loop of the TSHR (9). In a family with hereditary nonautoimmune hyperthyroidism harboring TSHR-D617Y, hyperthyroidism in the affected members

4 Copyright 2018 AACE Nonautoimmune Hyperthyroidism, AACE Clinical Case Rep. 2018;4(No. 1) e87 Fig. 2. Sequential clinical and laboratory findings during methimazole (MMI) treatment. FT3 = free triiodothyronine; FT4 = free thyroxine; TRAb = thyrotropin-receptor antibody; TSAb = thyroid-stimulating antibody; TSH = thyrotropin; US = ultrasonography. A B C D E Fig. 3. Imaging study and histopathology of the thyroid gland. Ultrasonography shows that multiple solid nodules are present in both lobes of the thyroid gland (A). The corresponding region of radioiodine uptake represents cold nodules, indicated by arrows (B). The resected thyroid tissue weighed 131g (C). Histopathologic features of the right lobe showing an encapsulated follicular adenoma (D*) and nonnodular parenchyma (D# and E).

5 e88 Nonautoimmune Hyperthyroidism, AACE Clinical Case Rep. 2018;4(No. 1) Copyright 2018 AACE Fig. 4. Thyrotropin receptor (TSHR) gene analysis. Pedigree of the investigated family. The arrow shows the proband in this study. Sequencing analysis of TSHR exon 10 in genomic DNA extracted from peripheral blood leukocytes. A guanine to adenine transition at position 929 (indicated by arrows; reverse sequence shown) was identified as a heterozygote in the patient and as a homozygote in his mother, and a wild-type sequence was detected in his father. A heterozygous adenine to guanine transition at position 1,856 (indicated by arrows; forward sequence shown) was identified in the patient, and wild-type sequences were detected in his parents. was well controlled by a small dose of MMI or inorganic iodine with mild goiter sizes ranging up to 34 ml, and the presentation of hyperthyroidism was delayed over 20 years (6,9). While this patient had no need for controlling hyperthyroidism until the age of 13 years, the long-term followup with MMI treatment showed further thyroid enlargement and nodular formation. In toxic adenomas, a mild increase in proliferation both with and without TSHR mutation and a decreased rate of apoptosis have been reported (10,11). Although thyroid size is variable in nonautoimmune hyperthyroidism, a comparative gene expression analysis showed that increased thyroid size caused by constitutive TSHR activation is not because of an increased proliferation rate but rather a decrease in apoptosis (12). In addition, an initially homogenous goiter tends to evolve into a multinodular goiter without malignancy (1). In this patient, benign thyroid nodules were detected using ultrasonography at the age of 17 years, and he underwent total thyroidectomy at the age of 25 years. Among 7 patients with sporadic nonautoimmune hyperthyroidism who were followed for more than 10 years after birth, 5 patients developed multinodular goiter at the age of 3 to 19 years and underwent near-total or total thyroidectomy (4,13-18). All TSHR germline mutations in sporadic nonautoimmune hyperthyroidism, including TSHR-D619G, are shared with somatic mutations found in toxic adenomas (5). However, interestingly, thyroid nodules were cold in this patient and in another patient with nonautoimmune hyperthyroidism (19), which suggests a loss of autonomy of the nodules. CONCLUSION In summary, we identified a Japanese patient with sporadic nonautoimmune hyperthyroidism caused by a TSHR mutation (D619G). While one elder patient with TSHR-N670S showed subclinical hyperthyroidism with large goiter and no family history of hyperthyroidism, information about her parents was lacking (20). To our knowledge, there are no other reports of sporadic cases of mild congenital hyperthyroidism that required no treatment until adolescence. The main reason is that the TSHR-

6 Copyright 2018 AACE Nonautoimmune Hyperthyroidism, AACE Clinical Case Rep. 2018;4(No. 1) e89 D619G mutation leads to a relatively smaller increase in constitutive activity than other TSHR mutations in sporadic nonautoimmune hyperthyroidism. Meanwhile, there was no remission by an antithyroid drug, and goiter developed with nodular formation. Therefore, it is important to perform ablative therapy without delay after the diagnosis. DISCLOSURE The authors have no multiplicity of interest to disclose. REFERENCES 1. Hébrant A, van Staveren WC, Maenhaut C, Dumont JE, Lèclere J. Genetic hyperthyroidism: hyperthyroidism due to activating TSHR mutations. Eur J Endocrinol. 2011;164: Aycan Z, Ağladioğlu SY, Ceylaner S, Cetinkaya S, Bas VN, Kendirici HN. Sporadic nonautoimmune neonatal hyperthyroidism due to A623V germline mutation in the thyrotropin receptor gene. J Clin Res Pediatr Endocrinol. 2010;2: Agretti P, De Marco G, Biagioni M, et al. Sporadic congenital nonautoimmune hyperthyroidism caused by P639S mutation in thyrotropin receptor gene. Eur J Pediatr. 2012;171: Nishihara E, Fukata S, Hishinuma A, et al. Sporadic congenital hyperthyroidism due to a germline mutation in the thyrotropin receptor gene (Leu 512 Gln) in a Japanese patient. Endocr J. 2006;53: Lueblinghoff J, Eszlinger M, Jaeschke H, et al. Shared sporadic and somatic thyrotropin receptor mutations display more active in vitro activities than familial thyrotropin receptor mutations. Thyroid. 2011;21: Nishihara E, Fukata S, Hishinuma A, Amino N, Miyauchi A. Prevalence of thyrotropin receptor germline mutations and clinical courses in 89 hyperthyroid patients with diffuse goiter and negative anti-thyrotropin receptor antibodies. Thyroid. 2014;24: Parma J, Duprez L, Van Sande J, et al. Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature. 1993;365: Palos-Paz F, Perez-Guerra O, Cameselle-Teijeiro J, et al. Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine-deficient area in NW Spain. Eur J Endocrinol. 2008;159: Nishihara E, Nagayama Y, Amino N, et al. A novel thyrotropin receptor germline mutation (Asp617Tyr) causing hereditary hyperthyroidism. Endocr J. 2007;54: Krohn K, Emmrich P, Ott N, Paschke R. Increased thyroid epithelial cell proliferation in toxic thyroid nodules. Thyroid. 1999;9: Deleu S, Allory Y, Radulescu A, et al. Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology. Thyroid. 2000;10: Hébrant A, Van Sande J, Roger PP, et al. Thyroid gene expression in familial nonautoimmune hyperthyroidism shows common characteristics with hyperfunctioning autonomous adenomas. J Clin Endocrinol Metab. 2009;94: Kopp P, van Sande J, Parma J, et al. Brief report: congenital hyperthyroidism caused by a mutation in the thyrotropin-receptor gene. N Engl J Med. 1995;332: Kopp P, Jameson JL, Roe TF. Congenital nonautoimmune hyperthyroidism in a nonidentical twin caused by a sporadic germline mutation in the thyrotropin receptor gene. Thyroid. 1997;7: Lavard L, Sehested A, Brock Jacobsen B, et al. Long-term follow-up of an infant with thyrotoxicosis due to germline mutation of the TSH receptor gene (Met453Thr). Horm Res. 1999;51: Führer D, Mix M, Wonerow P, Richter I, Willgerodt H, Paschke R. Variable phenotype associated with Ser505Asn-activating thyrotropin-receptor germline mutation. Thyroid. 1999;9: Bircan R, Miehle K, Mladenova G, et al. Multiple relapses of hyperthyroidism after thyroid surgeries in a patient with long-term follow-up of sporadic nonautoimmune hyperthyroidism. Exp Clin Endocrinol Diabetes. 2008;116: Bertalan R, Sallai A, Sólyom J, et al. Hyperthyroidism caused by a germline activating mutation of the thyrotropin receptor gene: difficulties in diagnosis and therapy. Thyroid. 2010;20: Nishihara E, Chen C, Higashiyama T, et al. Subclinical nonautoimmune hyperthyroidism in a family segregates with a thyrotropin receptor mutation with weakly increased constitutive activity. Thyroid. 2010;20: Schaarschmidt J, Paschke S, Özerden M, et al. Late manifestation of subclinical hyperthyroidism after goitrogenesis in an index patient with a N670S TSH receptor germline mutation masquerading as TSH receptor antibody negative Graves disease. Horm Metab Res. 2012;44: