THYROID ORIGINAL STUDIES

Volume 24, Number 5, 2014

ª Mary Ann Liebert, Inc. THYROID FUNCTION AND DYSFUNCTION
DOI: 10.1089/thy.2013.0431

Prevalence of Thyrotropin Receptor Germline Mutations

and Clinical Courses in 89 Hyperthyroid Patients
with Diffuse Goiter and Negative Anti-Thyrotropin
Receptor Antibodies

Eijun Nishihara,1 Shuji Fukata,2 Akira Hishinuma,3 Nobuyuki Amino,1 and Akira Miyauchi1

Background: We studied the frequency of thyrotropin (TSH) receptor mutations in hyperthyroid patients with
diffuse goiter and negative TSH receptor antibodies (TRAb), and the clinical pictures of the hyperthyroid
patients in the presence and absence of mutations.
Patients and Methods: From 2003 through 2012, 89 hyperthyroid patients with diffuse goiter and negative
TRAb based on a second- or third-generation assay underwent sequence analysis of the TSH receptor gene from
peripheral leukocytes. The outcome of hyperthyroidism in patients with a TSH receptor mutation and their
affected family members was compared with that in patients without any mutation after a 1–10-year follow-up.
Results: Germline mutations of the TSH receptor occurred in 4 of the 89 patients (4.5%), including 3 definitive
constitutively activating mutations (L512Q, E575K, and D617Y). The main difference in the clinical outcome
of hyperthyroidism was that no patients with a TSH receptor mutation achieved euthyroidism throughout the
follow-up, while 23.5% of patients without any mutation entered remission. The progression from subclinical to
overt hyperthyroidism was not significantly different between patients with or without a mutation. Meanwhile,
10.3% of TRAb-negative patients without any TSH receptor mutation developed TRAb-positive Graves’ hy-
perthyroidism during the follow-up.
Conclusions: The prevalence of nonautoimmune hyperthyroidism with TSH receptor mutations is lower than
that of latent Graves’ disease in TRAb-negative patients with hyperthyroidism. However, all affected patients
with a TSH receptor mutation showed persistent hyperthyroidism regardless of subclinical or overt hyperthy-
roidism throughout the follow-up.

Introduction (third generation) to the immobilized TSH receptor, but less

frequently by a bioassay to stimulate thyroid tissue (thyroid-

T he clinical diagnosis of patients with hyperthyroid-

ism is based on clinical manifestations and examination
findings regarding serum antithyroid antibodies, radioactive
iodine scintigraphy, and neck ultrasonography. Graves’ dis-
ease is the most prevalent among patients with hyperthyroid-
ism in areas where iodine is richly supplied, and the presence
of anti-thyrotropin (TSH) receptor antibodies is critical for its
diagnosis. In clinical practice, anti-TSH receptor antibodies
are detected using commercially available TSH receptor an-
tibody (TRAb) assays that measure the ability of the patient’s
serum to inhibit the binding of labeled TSH (second genera-
tion) or labeled monoclonal antibody against the TSH receptor
stimulating antibody, TSAb). Recent second- or third-generation
TRAb assays have demonstrated marked sensitivity for di-
agnosing Graves’ disease, ranging between 90% and nearly
100% depending on the cutoff value used (1,2).
Among disease categories of hyperthyroidism that present
with negative TRAb and diffuse goiter, nonautoimmune hy-
perthyroidism and a subset of toxic multinodular goiters with
homogeneous distribution of the tracer on thyroid scintigraphy
are known. Constitutively activating germline mutations of
the TSH receptor (TSHR) gene have been identified as a mo-
lecular cause of nonautoimmune hyperthyroidism, while
constitutively activating somatic mutations of the TSHR gene

1
Center for Excellence in Thyroid Care, Kuma Hospital, Kobe, Japan.
2
Tajiri Clinic, Kumamoto, Japan.
3
Department of Infection Control and Clinical Laboratory Medicine, Dokkyo Medical University, Tochigi, Japan.

789
790
are detected in approximately half of toxic multinodular goi-
ters. Serum TRAb levels gradually decrease in 80% of patients
with Graves’ disease during treatment with antithyroid drugs,
and are often undetectable at the remission stage (3,4). In
contrast, there are a small proportion of patients with Graves’
disease in whom TRAb become positive after treatment with
antithyroid drugs (5). Therefore, these patients with latent
Graves’ disease may also present with negative TRAb during
their clinical courses.
In nonautoimmune hyperthyroidism, the severity of hy-
perthyroidism and goiter size are variable, even among
family members harboring the same mutation. The absence
of autoimmune parameters may be a major clue for identi-
fying possible patients with nonautoimmune hyperthyroid-
ism. However, the prevalence of germline mutations of the
TSH receptor among hyperthyroid patients with negative
TRAb and diffuse goiter is uncertain. Here, we first studied
the frequency of germline mutations of the TSHR gene in
patients described above. Then, the clinical pictures of hy-
perthyroidism were compared in patients with and without
TSH receptor mutations.
Patients and Methods
Patients
From 2003 through 2012, in total 24,623 patients with
hyperthyroidism were initially referred to Kuma Hospital. In
subjects with subclinical or overt hyperthyroidism, we en-
countered 89 patients (13 men and 76 women; aged 40 – 12.5
years [median – quartile deviation]; 2–79 years [range]) who
were unlikely to have Graves’ disease or toxic multinodular
goiter based on the clinical data shown in Table 1. The age of
the patients at the diagnosis of hyperthyroidism was 34 – 11
years. Sequence analysis of the TSHR gene was carried out
for these patients, after an interval of 2 – 2 years. When a
germline mutation was detected in a patient, a family screening
test for the mutation was carried out involving all available
family members (n = 15, aged 38 – 12.5 years; Fig. 1). The
medical history, first-degree family history of hyperthyroid-
ism, and presence of Graves’ ophthalmopathy were recorded.
The clinical courses of all affected patients with TSH receptor
mutations and those of patients without any mutation were
followed up.
Thyroid function analysis and thyroid volume
Concentrations of serum TSH, free triiodothyronine (FT3),
and free thyroxine (FT4) were measured with a chemilumi-
Table 1. Clinical Pictures of Hyperthyroid Patients with Diffuse Goiter
and Negative Thyrotropin Receptor Antibodies in This Study
Number of
patients Hyperthyroidism TRAb RAIU
65 Overt or subclinical Negative High and diffuse uptake
12 Subclinicala Negativeb NT
12 Overta Negativeb NT
a
Persistent hyperthyroidism for more than 1 year.
b
No history of positive findings.
ATD, antithyroid drugs; NT, not tested; RAIU, thyroidal radioactive iodine uptake; TRAb, TSH receptor antibodies; TSH, thyrotropin;
US, ultrasonographic findings of the thyroid gland.
NISHIHARA ET AL.
nescent immunoassay (Architect i2000; Abbot Japan, Tokyo,
Japan). The reference ranges used for serum TSH, FT3, and
FT4 were 0.30–5.00 lIU/mL, 1.70–3.70 pg/mL, and 0.70–
1.60 ng/dL, respectively. Subclinical hyperthyroidism was
diagnosed based on a suppressed TSH with normal FT4 and
FT3 levels. Overt hyperthyroidism was diagnosed based on
high FT4 and/or FT3 levels combined with a suppressed
TSH. The thyroid function of patients receiving continuous
administration of antithyroid drugs or who had already un-
dergone ablative therapies such as radioactive iodine and
thyroidectomy was also defined as overt hyperthyroidism.
Serum TRAb were measured with a second-generation
enzyme-linked immunoassay (RSR Ltd., Cardiff, United
Kingdom) through July 2008 and with a third-generation
electrochemiluminescent immunoassay (Roche Diagnostic,
Mannheim, Germany) from August 2008 through December
2012. Serum TSAb were measured with a commercial bio-
assay kit (Yamasa Co., Chiba, Japan). The upper cutoff limits
of values of the second-generation TRAb, third-generation
TRAb, and TSAb are 15%, 1.9 IU/L, and 180%, respectively.
Serum anti-thyroglobulin antibodies (TgAb) and anti-thyroid
peroxidase antibodies (TPOAb) were measured with an
electrochemiluminescent immunoassay (ECLusys Anti-Tg
and ECLusys Anti-TPO; Roche Diagnostic). The upper cut-
off limits of values of TgAb and TPOAb were 39.9 and
27.9 U/mL, respectively. The total thyroid volume was
measured using ultrasonography, as reported previously (6).
DNA sequencing
Genomic DNA was extracted from peripheral leukocytes
with GenTLE (Takara, Kyoto, Japan). Exons 9 and 10 of the
TSHR gene encoding the entire intracellular and transmem-
brane regions and part of the proximal extracellular domain
of the TSH receptor were amplified by the polymerase chain
reaction (PCR) using High Fidelity PCR Master (Roche Di-
agnostic), as described previously (7). Direct sequencing of
PCR products was performed using the Bigdye Terminator
v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City,
CA) and an automatic ABI 3130 sequencer (Applied Bio-
systems). The present study was approved by the ethics
committee of Kuma Hospital, and informed consent was
obtained from the patients and their family members for the
use of samples for research purposes.
Statistical analysis
Thyroid volumes were compared using the Mann–Whitney
test. The first-degree family history of hyperthyroidism,
Number of patients with
US Therapy TSH receptor mutation
Diffuse None 3
Diffuse None 0
Diffuse On ATD 1
TSH RECEPTOR MUTATIONS IN HYPERTHYROID PATIENTS 791

FIG. 1. Flow chart of

the study population. TSH,
thyrotropin.

presence of TgAb and/or TPOAb, stable state of subclinical
hyperthyroidism, remission to euthyroidism, and therapeu-
tic modalities were compared between the two groups with
the v2-test. p < 0.05 was accepted as indicating significance.
Results
Sequence analysis of the TSHR gene of the 89 patients led
to the identification of 4 mutations in 4 patients (L512Q,
E575K, D617Y, and L267F; Table 2). The four patients were
heterozygous. The family screening test for each mutation
identified two additional patients harboring E575K and five
patients harboring D617Y (Figs. 1 and 2). Eleven patients
with TSH receptor mutations presented with subclinical or
overt hyperthyroidism, but the remaining family members
without any TSH receptor mutation were euthyroid. The
mutations of L512Q, E575K, and D617Y were confirmed as
constitutively active forms by in vitro functional assays (6,8–
10). The proband with L512Q was a de novo case without the
presence of a mutation in her parents (Fig. 2A), and had
inadequate treatment for congenital hyperthyroidism, which
caused bone abnormalities and mental retardation (8). Other
probands with a TSH receptor mutation had a first-degree
family history of hyperthyroidism (Fig. 2B–D). The in vitro
activity of L267F was not tested. The proband’s mother had a
history of hyperthyroidism but had already died without de-
tailed information. Clinical pictures of patients with TSH
receptor mutation were evaluated over a 3–10-year follow-up

Table 2. Clinical Characteristics of 11 Patients with Thyrotropin Receptor Mutations

First visit Follow-up
Age Goiter Period Goiter
Mutation (years) Sex (mL) Hyperthyroidism Therapy (years) (mL) Hyperthyroidism Therapy TgAb/TPOAb
L267F 76 F 35 Overt None 3 18 Overt ATD -/-
(801G > T)
L512Q 20 F 370 Overt On ATD 10 181 Overt RI/ATD -/-
(1535T > A)
E575Ka 61b F 40 Subclinical None 9 45 Subclinical None +/+
(1723G > A) 38 M 39 Subclinical None 5 31 Subclinical None -/-
30 M 36 Subclinical None 5 37 Subclinical None -/-
D617Ya 55 F 11 Subclinical None 8 18 Subclinical None -/-
(1849G > T) 48 F 59 Overt On ATD 8 NT Overt Surgery -/-
24 F 28 Subclinical None 8 49 Overt ATD -/+
21 F 22 Overt None 8 19 Overt KI -/-
20b F 35 Overt None 8 7.4 Overt RI -/-
20 M 12 Subclinical None 8 14 Subclinical None -/-
a
Mutation affects several members of the same family.
b
Proband.
Age, age at diagnosis; F, female; KI, potassium iodide; M, male; RI, radioactive iodine therapy; TgAB, anti-thyroglobulin antibodies;
TPOAb, anti-thyroid peroxidase antibodies.
792 NISHIHARA ET AL.

FIG. 2. The family pedigrees of patients

with TSH receptor mutations. (A) L512Q;
(B) E575K; (C) D617Y; (D) L267F. Arrows
indicate the probands. Sequence analysis
of the TSH receptor gene was carried out
for the probands and their family mem-
bers designated by asterisks.

(Table 2). In 6 patients with subclinical hyperthyroidism at
the first examination, 5 patients maintained a stable state of
subclinical hyperthyroidism without increasing goiter size,
but 1 patient progressed to overt hyperthyroidism at the age
of 25 years. Five patients with overt hyperthyroidism at the
first examination were treated with continuous medication or
thyroid ablation. No patients achieved euthyroidism without
treatment throughout their clinical courses. TgAb and/or
TPOAb were detected in 2 of 11 patients with a mutation
(18.2%; Table 2).
In 85 patients without any TSH receptor mutation, clinical
follow-up data on 68 patients were available for more than 1
year (median 4.0 years; Fig. 1). TgAb and/or TPOAb were
detected in 17 of the 68 patients (25.0%). Throughout their
clinical courses, 7 of the 68 TRAb-negative patients (10.3%)
developed TRAb-positive Graves’ hyperthyroidism. There-
after, 2 of the 7 patients entered remission within 3 years of
antithyroid drug therapy (Table 3). Among 61 patients with
persistently negative TRAb, 39 patients (63.9%) were sub-
clinical hyperthyroid or euthyroid. Of 34 patients with sub-
clinical hyperthyroidism at the first examination, 22 (64.7%)
patients maintained a stable state of subclinical hyperthy-
roidism, while 6 patients (17.6%) received medication or
underwent ablative therapy, and 6 patients (17.6%) entered
remission. Fifty-four of the 61 patients with persistently
negative TRAb were tested by the TSAb assay and then 5
patients (9.3%) showed weakly positive TSAb. Among them,
four patients entered remission and one patient underwent
total thyroidectomy. Subsequently, histopathological exam-
ination of this thyroid specimen showed follicular swelling,
papillary projection, vacuolated colloid, and lymphocytic
infiltration in the stroma, which were compatible with
Graves’ disease. Histopathological findings of thyroid spec-
imens from six other patients negative for both TRAb and

Table 3. Outcome of 68 Patients Without Any Thyrotropin Receptor Mutation

After More Than One-Year Follow-Up
First visit Follow-up
Hyperthyroidism TRAb Thyroid function Therapy Period (years)
Overt [5] Positive [7] Overt hyperthyroid [5] ATD [3] 2–5
Subclinical [2] RI [1]
Surgery [1]
Euthyroid [2] None 3–4
Overt [27] Negative [61] Overt hyperthyroid [22] ATD [8] 1–10
Subclinical [34] KI [2]
RI [5]
Surgery [7]
Subclinical hyperthyroid [25] None 1–10
Euthyroid [14] None 1–7
The brackets indicate numbers of patients.
TSH RECEPTOR MUTATIONS IN HYPERTHYROID PATIENTS
FIG. 3. Comparison of the clinical characteristics of patients with or without TSH receptor mutations. Seven patients with
a TSH receptor mutation identified in the separate family study were added to the initial 89 patients. (A) Age distribution at
diagnosis. (B) Thyroid volume at diagnosis. Horizontal lines indicate median values.
TSAb (Table 3) showed multiple adenomatous nodules but
no lymphocytic infiltration in the thyroid gland.
In our study, all subjects were over 19 years old, excluding
2 patients (2 and 6 years old) without any TSH receptor
mutation (Fig. 3A). Hyperthyroidism in first-degree family
members was detected in 3 of 4 probands with a TSH receptor
mutation (75.0%), while it was detected in 26 of 85 patients
without any mutation (30.6%; v2 = 3.43, p = 0.06). The thy-
roid volume at the first examination was not significantly
different between patients with and without a TSH receptor
mutation (Fig. 3B). Therapeutic modalities with no treat-
ment, medication, and ablative therapy for hyperthyroidism
were not significantly different between these two groups
(Tables 2 and 3; v2 = 2.65, p = 0.27). No patients presented
with Graves’ ophthalmopathy.
Discussion
The presence of TSH receptor mutations is the primary
cause of nonautoimmune hyperthyroidism. In this study, the
prevalence of a TSH receptor mutation was 4.5% in 89 hy-
perthyroid patients who were unlikely to have Graves’ dis-
ease or toxic multinodular goiter based on the clinical data
shown in Table 1. Previous studies concerning non-
autoimmune hyperthyroidism were mostly case reports, but
the prevalence among patients with hyperthyroidism has
been less extensively studied. To our knowledge, only a
country-wide screening in Denmark showed that the preva-
lence of nonautoimmune hyperthyroidism with constitutively
activating germline mutations of the TSHR gene was 0.8%
in 121 patients with juvenile-onset hyperthyroidism, which
corresponds to 5.8% in 17 patients with juvenile-onset hy-
perthyroidism who do not have thyroid autoantibodies, in-
cluding TSAb, TgAb, and TPOAb (11). In our study, subjects
over 19 years old, excluding 2 patients (Fig. 2A), seem to be an
appropriate population because hereditary nonautoimmune
hyperthyroidism develops most frequently during adolescence
793
or later (12). Sequence analysis of the TSHR gene in this
population gave us a chance to identify not only typical pa-
tients with hereditary nonautoimmune hyperthyroidism, but
also one patient with unnoticed sporadic nonautoimmune hy-
perthyroidism. Even in the presence of TgAb and/or TPOAb,
nonautoimmune hyperthyroidism can be differentiated from
autoimmune thyroid diseases, including painless thyroiditis
and the so-called Hashitoxicosis, based on the levels of ra-
dioactive iodine uptake and clinical follow-up of the thyroid
function. The presence of TgAb and/or TPOAb in patients is
not required to exclude the diagnosis of nonautoimmune hy-
perthyroidism (13–15). Indeed, 18.2% of the patients with a
TSH receptor mutation were concomitantly positive for TgAb
and/or TPOAb; the positive rate was not significantly different
from patients without any mutation (v2 = 0.24, p = 0.62).
In spite of the variable severity of hyperthyroidism among
patients with nonautoimmune hyperthyroidism, data from
previous reports show that affected adult patients develop
almost always overt hyperthyroidism and receive continuous
antithyroid drug therapy or ablative therapy (12). Indeed, a
20-year-old patient in this study was finally diagnosed with
sporadic nonautoimmune hyperthyroidism with an L512Q
mutation following inadequate treatment for congenital hy-
perthyroidism (8). Immediate diagnosis and initiation of
ablative therapy without delay is indispensable to avoid
irreversible complications (16–23). TSH receptor mutations
in sporadic cases, including our case, typically have a more
severe clinical presentation and much stronger activating
effect than those in hereditary cases, which has also been
shown by in vitro studies (24). In contrast, 5 of 10 adult
patients with hereditary nonautoimmune hyperthyroidism
maintained a stable state of subclinical hyperthyroidism
without treatment for more than 8 years, which may be de-
pendent on the relatively small increase in constitutive activity
of TSH receptor mutations (E575K and D617Y) and other
environmental modifiers, including iodine intake (6,7,10).
These affected patients with persistently subclinical
794
hyperthyroidism showed no symptoms of hyperthyroidism or
complications of cardiovascular diseases such as atrial fibril-
lation. In our study, more than 60% of patients with subclinical
hyperthyroidism did not develop overt hyperthyroidism with
either the presence or absence of a TSH receptor mutation
(v2 = 0.81, p = 0.37). Therefore, these patients with mild hy-
perthyroidism cannot be accurately classified without molec-
ular analysis of the TSHR gene. No information about in vitro
activity of L267F has been obtained yet. L267 is located in the
amino-terminal hinge region of TSH receptor ectodomain,
where both mechanisms of hormone binding and intramo-
lecular signal transduction toward the serpentine domain are
assumed, and naturally occurring pathogenic activating mu-
tations have been reported (25,26).
At the first examination, there were no significant differ-
ences in clinical data between patients with and without a
TSH receptor mutation. When compared with the thyroid
function during follow-up, 23.5% of patients without any
TSH receptor mutation entered remission (Table 3), but no
patients with a mutation achieved euthyroidism throughout
the follow-up (Table 2; v2 = 3.25, p = 0.07). Although sig-
nificance was not noted, persistent hyperthyroidism rather
than hormonal levels at the onset may lead to characteristic
features in nonautoimmune hyperthyroidism. While a diag-
nosis of Graves’ disease depends on the presence of TRAb,
approximately 10% of initially TRAb-negative patients
without any TSH receptor mutation turned out to be TRAb
positive during the follow-up (Table 3), and 9.3% of patients
with persistently negative TRAb were weakly positive for
TSAb in our study. The sensitivity of TRAb to diagnose
Graves’ disease is high but not 100%, especially at its early
stage with mild hyperthyroidism or near remission during
antithyroid drug treatment (3–5,27). When considering all
patients with positive TRAb or TSAb during the follow-up,
or who entered remission without treatment, at least 22 of the
68 patients (32%) without any TSH receptor mutation are
considered to have had latent Graves’ disease at the first
examination. On the basis of periodic follow-up combined
with several examinations in persistent TRAb-negative pa-
tients, however, the exact etiology of hyperthyroidism re-
mains undefined in more than half of the patients. There is a
considerable interindividual variation of the set point of TSH
in healthy subjects, which is determined by genetic and en-
vironmental factors. Accordingly, a suppressed TSH with
normal FT4 and FT3 levels may not always be identical with
subclinical hyperthyroidism.
In summary, we focused on patients with subclinical or
overt hyperthyroidism with diffuse goiter and negative TRAb
in the second- or third-generation assay. In this condition, the
prevalence of germline mutations of the TSHR gene is 4.5%.
All patients with a TSH receptor mutation were adults at the
time of genetic testing and showed persistent hyperthyroidism
regardless of the presence of subclinical or overt hyperthy-
roidism. To address the long-term care of hyperthyroid patients
and their affected family members, we should evaluate TSH
receptor mutations in persistently TRAb-negative patients with
hyperthyroidism, not only in children, but also in adults.
Acknowledgments
We are grateful to Drs. Yuji Nagayama and Basil Rapoport
for their excellent in vitro studies to elucidate the constitutive
NISHIHARA ET AL.
activity concerning D617Y and E575K mutations, respec-
tively, cited herein, as well as to Kuma Hospital staff for their
invaluable contribution.
Author Disclosure Statement
The authors declare that no competing financial interests
exist.
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Address correspondence to:
Eijun Nishihara, MD, PhD
Center for Excellence in Thyroid Care
Kuma Hospital
8-2-35 Shimoyamate-dori
Chuo-ku, Kobe 650-0011
Japan
E-mail: nishihara@kuma-h.or.jp