Molecular pathogenesis of euthyroid and toxic multinodular goiter

Endocrine Reviews. Published December 22, 2004 as doi:10.1210/er.2004-0005 

Molecular pathogenesis of euthyroid and toxic 

multinodular goiter 

KNUT KROHN 1,2 , DAGMAR FÜHRER 1 , YVONNE BAYER 1 , MARKUS ESZLINGER 

1 , VOLKER BRAUER 1 , SUSANNE NEUMANN 1 AND RALF PASCHKE 1 

1 III. Medical Department, University of Leipzig 

Ph.-Rosenthal-Str. 27, D-04103 Leipzig, Germany 

2 Interdisciplinary Center for Clinical Research Leipzig, University of Leipzig 

Inselstraße 22, D-04103 Leipzig, Germany 

Correspondence: 

R. Paschke, M.D. 

III. Medical Department, University of Leipzig 

Ph.-Rosenthal-Str. 27, D-04103 Leipzig, Germany 

Phone: 49-341-9713200 

Fax: 49-341-9713209 

email: pasr@medizin.uni-leipzig.de 

Copyright (C) 2004 by The Endocrine Society

Summary 

The purpose of this review is to summarize the current knowledge of the etiology of 

euthyroid and toxic multinodular goiter with respect to the epidemiology, clinical 

characteristics and molecular pathology. 

In reconstructing the line of events from early thyroid hyperplasia to multinodular goiter we 

will argue the predominant neoplastic character of nodular structures, the nature of known 

somatic mutations and the importance of mutagenesis. Furthermore, we outline direct and 

indirect consequences of these somatic mutations for thyroid pathophysiology and summarize 

information concerning a possible genetic backround of euthyroid goiter. 

Finally we discuss uncertainties/open questions in differential diagnosis and therapy of 

euthyroid and toxic multinodular goiter. 

I Definition and Epidemiology 

II Clinical aspects of euthyroid and toxic multinodular goiter 

A Differential diagnosis 

III Natural course of euthyroid and toxic multinodular goiter 

A Nodule Growth 

B Thyroid function 

IV Clonal origin of thyroid nodules 

V Hot thyroid nodules 

A Signal transduction of HTN with and without TSHR mutations 

B Secondary/indirect effects of activating TSHR mutations 

VI Cold thyroid nodules 

A Iodide transport and metabolism 

B Signaling proteins 

C Results of gene expression studies by arrays 

D Chromosomal aberrations 

VII Multinodular Goiter 

A Mutagenesis as the cause of nodular transformation 

B Etiology 

VIII Pathogenesis and genetic etiology of euthyroid goiter 

A Family and twin studies 

B Candidate loci 

C Linkage analysis 

IX Perspectives 

A Therapeutic implications 

B Diagnostic implications

I Definition and Epidemiology 

Benign nodular thyroid disease constitutes a heterogenous thyroid disorder, which is highly 

prevalent in iodine deficient areas. On a very general basis it can be divided into solitary 

nodular and multinodular thyroid disease. Histologically, benign thyroid nodules are 

distinguished as 1. encapsulated lesions (true adenomas) or adenomatous nodules, which lack 

a capsule, and 2. by morphological criteria according to the WHO classification (1). On 

functional grounds, nodules are classified as either “cold”, “normal” or “hot” depending on 

whether they show decreased, normal or increased uptake on scintiscan. Approximately 85% 

of all nodules are “cold”, 10% are normal and 5% are “hot” (2;3), although the prevalence 

may vary geographically with the ambient iodine supply. In contrast to solitary nodular 

thyroid disease, which has a more uniform clinical, pathological and molecular picture, 

euthyroid multinodular goiter (MNG) and toxic multinodular goiter (TMNG) are a mixed 

group of nodular entities, i.e. one usually finds a combination of hyper-, hypofunctional or 

normally functioning thyroid lesions within the same thyroid gland. The overall balance of 

functional properties of individual thyroid nodules within a multinodular goiter ultimately 

determine the functional status in the individual patient, which may be euthyroidism (normal 

TSH and free thyroid hormone levels), subclinical hyperthyroidism (low or suppressed TSH 

and normal free thyroid hormone levels) or overt hyperthyroidism (suppressed TSH and 

elevated free thyroid hormone levels). The term MNG is applied in the first scenario while 

TMNG refers to the latter situations. It is important to emphasize that this functional picture is 

not stationary but patients with TMNG usually have a history of long-standing MNG (4). 

Moreover, the status of TSH suppression in TMNG does not only imply clinical consequences 

for the patient but importantly also indicates that a critical level of thyroid autonomy i.e. 

independence of thyrotropin (TSH), the physiological regulator of thyroid function and 

growth (5;6) has been reached. Constitutive activation of the cAMP signaling pathway is 

widely accepted as the biochemical driving force of thyroid autonomy as suggested by the 

3

presence of somatic activating TSH receptor (TSHR) mutations in scintigraphically non- 

suppressible foci in euthyroid goiters in iodine deficient areas, the presence of somatic TSHR 

mutations and less frequently Gs alpha protein mutations in macroscopic toxic thyroid 

nodules both in solitary nodules and multinodular disease, the phenotype of patients with 

activating germline TSHR mutations and a number of animal models of thyroid autonomy 

(reviewed in (7-10)). 

Iodine deficiency is by far the best studied epidemiologic risk factor for nodular thyroid 

disease: the prevalence of nodular thyroid disease (as well as goiter) is inversely correlated 

with the population’s iodine intake (11;12). This has formerly been assessed clinically by 

palpation, nowadays considered highly inaccurate (13-15), but is also clearly documented by 

thyroid ultrasonography. Based on ultrasound investigation a frequency of thyroid nodular 

disease as high as 30-40% (women) and 20-30% (men) of the adult population has been 

reported in iodine deficient areas. Furthermore even minor differences in the ambient iodine 

supply may be reflected in the different prevalence of thyroid abnormalities: Knudsen et al. 

(16) found a difference in goiter prevalence (15% in mild and 22.6% in moderate deficiency) 

and nodule size (increased in the moderate iodine deficiency group). The prevalence of 

thyroid nodules seems to increase with age (4;17;18). In a borderline iodine deficiency area 

MNG was present in 23% of the studied population of 2656 Danish people aged 41 to 71 yr., 

and increased with age in women (20 to 46%) as well as men (7 to 23%) (3). In contrast, the 

relation between age and thyroid volume is less coherent, whereby in iodine deficient areas 

(except for severe deficiency), thyroid enlargement peaks around 40 years with no tendency 

for further increase (19). Interestingly, similar observations have been made in an iodine 

sufficient area. In the twenty year follow-up of the Whickham Survey the frequency of goiter 

decreased with age (goiter prevalence initially: 23% women and 5% men; at 20 year follow- 

up of the same patients: 10% women and 2% men) (20). 

4

Thyroid nodules are found with higher frequency in enlarged thyroid glands though all 

clinicians will agree that they may also be present in an otherwise normal thyroid gland. 

(4;18;21). The correlation between iodine supply and prevalence of nodular thyroid disease 

can similarly also applies to toxic multinodular goiter. The high frequency of thyroid 

autonomy, which accounts for up to 60% of cases of thyrotoxicosis in iodine deficient areas is 

largely due to TMNG (~ 50%, solitary toxic nodules ~ 10%) (12;22). Prevalence of thyroid 

autonomy correlates with increased thyroid nodularity and increases with age (4;22). In 

contrast, thyroid autonomy is rare (3-10% of cases of thyrotoxicosis) in regions with 

sufficient iodine supply (22;23;23). Correction of iodine deficiency in a population results in 

decrease of thyroid autonomy as demonstrated by the impressive 73% reduction in prevalence 

of TMNG only 15 yr. after the doubling of iodine content of salt in Switzerland (12;24). 

While goiter and euthyroid and toxic nodular thyroid disease share the common and important 

epidemiology of iodine deficiency (ID), it needs to be stressed that most epidemiological 

conclusions are derived from cross sectional studies.Thyroid nodules (and goiter) also occur 

in individuals without exposure to iodide deficiency and not all individuals in an iodine 

deficient region develop a goiter . Moreover there is a strong clustering of goiter in families 

(see chapter VIII). 

Screening has been performed for other “environmental factors” (19). Smoking has been 

proposed as a risk factor for goiter (25) and nodules were also found with higher prevalence 

in goiters of smokers compared to non-smokers. The impact of smoking on thyroid disease 

could be due to i.e. increased thiocyanate levels in smokers exerting a competitive inhibitory 

effect on iodide uptake and organification (19;26). The association is more pronounced, again, 

in iodine deficiency (26). Radiation is another environmental risk factor not only for thyroid 

malignancy but also for benign nodular thyroid disease. An increased prevalence of nodular 

disease has been associated with exposure to radionuclear fallouts and therapeutic external 

radiation and is discussed by some authors also for occupational exposure to low-level 

5

adiation (27-31). Furthermore several studies suggest that thyroid volume is also 

significantly correlated with body weight and body mass index. In agreement with this a 

recent study has shown that in obese women weight loss of more than 10% may result in a 

significant decrease in thyroid volume (32). 

Nodular disease is more frequent (5-15 fold (33;34)) in women and yet the reasons for this are 

poorly understood. Thus at present one can only speculate as to a genetic susceptibility for 

thyroid disease (for details see chapter VIII) and/or a direct impact of steroid hormones. 

In fact, a growth promoting effect of estrogen has been described in vitro in rat FRTL-5 cells 

and thyroid cancer cell lines and has been proposed as a possible contributing, constitutional 

effect of gender (35;36). In addition, 17β- estradiol has been suggested to amplify growth 

factor induced signaling in normal thyroid and thyroid tumors (36). Interestingly, the use of 

oral contraceptives which antagonise the physiological hormonal cycle has been reported to 

be associated with a decrease in goiter (but not nodules even though this may represent an age 

artefact of the studied population). On the other hand pregnancy related thyroid enlargement 

was clearly related to iodine deficiency (19) and in one German study increased MNG 

prevalence with parity was only observed in those women which had not taken iodine 

supplementation during earlier pregnancy (37). 

In summary the development of nodular disease is influenced by multiple environmental 

components in interaction with constitutional parameters of gender and age. However whether 

these factors actually result in goiter or nodular thyroid disease is a different matter ultimately 

decided on the genetic background of the individual patient, discussed below (see chapter 

VIII). 

II Clinical aspects of euthyroid and toxic multinodular goiter 

6

Clinical features in a patient with multinodular goiter can be attributed to thyroid enlargement 

and thyrotoxicosis in case of TMNG. Thus, a patient may present with a lump or 

disfigurement of the neck, intolerance of tight necklaces or increase in collar size. Dysphagia 

or breathing difficulties due to local easophageal or tracheal compression may be apparent, 

especially with large goiter (33). Besides cosmetic aspects and compression signs, the daily 

challenge is to identify very rare thyroid malignancy in very frequent nodular thyroid disease 

and strategies to approach that goal have been reviewed in detail elsewhere (38-41). 

Alternatively, patients may present with symptoms suggestive of hyperthyroidism, the clinical 

presentation of which varies considerably with age. In a series of 84 French patients with 

overt hyperthyroidism, classical signs of thyrotoxicosis e.g. nervousness, weight loss despite 

increased appetite, palpitations, tremor and heat intolerance were more frequently observed in 

younger patients (≤ 50 years) (42), while atrial fibrillation and anorexia dominated in the 

older age group (≥ 70 yr.). In addition, subclinical hyperthyroidism, defined by low or 

suppressed TSH with normal fT4 and fT3 levels is more commonly observed in older patients 

with TMNG (43). In fact the incidental finding of low or suppressed TSH levels on routine 

investigation in iodine deficient regions for other conditions is frequently a first indicator for 

presence of thyroid autonomy (4). Subclinical hyperthyroidism is more than “just” a low TSH 

status, since it is associated with increased prevalence of atrial fibrillation and bone density 

loss (43). In addition, an increased cardiovascular mortality rate in patients with low serum 

TSH levels has been described in a 10 yr. cohort-study in the UK (44). The management of 

euthyroid and toxic multinodular thyroid disease has recently been extensively reviewed by 

Hegedüs and coworkers (33). 

A Differential diagnosis 

7

Very rarely TMNG occurs as an autosomal dominantly inherited disease caused by activating 

germline mutations in the TSHR gene (45). A positive family history of recurrent 

hyperthyroidism and goiter with absence of typical diagnostic features of Graves’ disease, 

persistent neonatal thyrotoxicosis and relapsing non-autoimmune thyrotoxicosis in childhood 

are highly suggestive of the condition. So far more than 150 patients (10 families and 11 

children with sporadic occurrence of TSHR germline mutations) have been reported in the 

literature (http://www.uni-leipzig.de/innere/TSHR). Thyroid ablation is advocated as the first 

line treatment (surgery and/or radioiodine) to prevent relapses. Molecular analysis for 

germline TSHR mutations offers the possibility for family screening, preclinical diagnosis 

and genetic counselling (7;46). 

In iodine deficient areas, the distinction between thyroid autonomy and Graves`disease (GD) 

can be complicated by absence of extrathyroidal signs of autoimmune thyroid disease and 

“atypical” diagnostic findings. In this regard several possibilities may be encountered. Firstly, 

erroneous classification of Graves disease as TMNG due to presence of thyroid nodules 

observed in 10-15% of GD patients or a patchy scintiscan appearance compatible with TMNG 

(47;48). Secondly, failure to detect TSHR antibodies (TRAB) in Graves’ disease using less 

sensitive assays. This is illustrated by the detection of TRAB with highly sensitive 2 nd 

generation assays and/or bioassays in up to 56% of patients with scintiscan appearance of 

TMNG and up to 22% of patients with diffuse uptake and absence of eye disease and negative 

TRAB results in older essays (hence erroneously classified as “diffuse thyroid autonomy”) 

(47-49). Thirdly, confusion of familial occurence of (autoimmune) hyperthyroidism with 

hereditary thyroid autonomy, which might clinically masquerade as Graves`disease. In this 

scenario, absence of TRAB is highly suggestive of familial non-autoimmune hyperthyroidism 

due to a constitutively activating TSHR germline mutation (46). 

8

III Natural course of euthyroid and toxic multinodular goiter 

A Nodule Growth 

From the epidemiological data discussed above one might expect an inherent progressive 

course of nodular thyroid disease. Studies aimed at accurate assessment of the nodules’ fate 

by ultrasonography differ in terms of follow-up period, definition of growth (increase in 

volume or nodule diameter), type of thyroid lesion (solid, cystic) and the background, in 

which they are conducted (e.g. environmental factors, specialised thyroid clinic). Moreover 

the inter-observer variability of long-term studies of nodule volumes is not known. With these 

caveats in mind the following observations have been reported: In iodine sufficient areas 

nodule “growth” has been reported in 35% of US patients over a follow-up period of 4.9 to 

5.6 yr (50). In another US study nodule growth (>15% increase in volume) was observed with 

similar frequency over a highly variable follow-up period (1 month to 5yr) (51). On long-term 

follow-up over 15 yr. in an area of iodine sufficiency only 1/3 of benign nodules showed 

growth as assessed by palpation and ultrasonography as opposed to the majority of nodules, 

which remained unchanged or even showed a decrease in size (52;53). In the German setting, 

for which the iodine deficit has been calculated at 30% of the recommended intake (54) a 

mean 3 yr. follow-up of 109 consecutive patients showed a steady and significant (> 30% 

volume) increase in nodular size in 50% of patients (55). In a Danish study only 4 (8%) of 45 

cold nodules in an area of borderline iodine deficiency showed a change in size (>5 mm in 

diameter) of which only 1 nodule actually increased whereas 3 nodules shrunk over a follow- 

up period of 2 yr. (table 1 (3)). The conclusion, which is suggested by these data is that both 

in an iodine deficient and sufficient setting a variable portion but most likely not all nodules 

will grow and the speed of growth is highly heterogeneous. Thus, identification of nodules 

with an increased growth potential is a challenge. This may also be relevant to the therapeutic 

management. In fact one could speculate that discrepant results reported in various treatment 

studies may actually reflect this heterogeneity of proliferation (and/or the potential to taper it 

9

down by treatment) rather than an evidence-based treatment effect of e.g. iodine vs. iodine 

plus l-thyroxine or l-thyroxine alone. Furthermore, results of currently available studies 

(3;52;53;55) do not allow conclusions as to whether nodule growth is associated with an 

increased risk of thyroid malignancy and thus the question arises as to the benefits of nodule 

volume reduction. In the authors’ opinion therapy, if efficient, is possibly better aimed at the 

primary prevention of the evolution of novel/further thyroid nodules in predisposed patients 

(56;57) with the long-term perspective to reduce ablative thyroid treatment for cosmetic 

reasons, compression symptoms and importantly thyroid malignancy. However, the proof of 

principle for any of these suggestions is (still) awaited. 

B Thyroid function 

Besides growth, transition from euthyroidism to hyperthyroidism in a patient with 

multinodular thyroid disease is an even more relevant clinical issue. We know that 

hyperthyroidism in TMNG develops insidiously and that TMNG is usually preceeded by a 

long-standing euthyroid multinodular goiter. In fact autonomous areas have been described in 

up to 40% of euthyroid goiters in iodine deficient regions (58). The most accurate 

epidemiological data on evolution of hyperthyroidism have been published for solitary toxic 

adenoma and most of these aspects can possibly also apply to MNG. The natural course is 

slow. An overall 4.1% annual incidence of thyrotoxicosis was observed in a group of 375 

untreated euthyroid patients with toxic adenoma (TA) in Germany, who were followed for a 

mean period of 53 months (59). In two longitudinal studies an incidence of 9-10% of overt 

thyrotoxicosis has been reported in patients with euthyroid MNG over a mean follow-up 

period of up to 12.2 years (60;61). There is a correlation between nodule size and 

development of hyperthyroidism: In an American study 93.5% of patients with overt 

hyperthyroidism had TA > 3cm in size and patients with a euthyroid TA of > 3cm size carried 

10

a 20% risk of developing hyperthyroidism during a 6yr. follow-up period as opposed to a 2- 

5% risk of patients with nodules < 2.5cm in size (23). Similarly in areas with iodine 

deficiency an autonomous volume of 16 ml has been determined to be critical for clinical 

manifestation of hyperthyroidism (62). In TMNG the extent of thyroid nodularity (and hence 

the autonomous volume) is related to the prevalence of low or suppressed TSH levels and 

both parameters are correlated with age (4). A sudden stimulation of thyroid function 

resulting in clinical manifestation as thyroid autonomy can be induced by the administration 

of excessive amounts of iodine e.g. in form of contrast media widely used for angiography 

and CT scans or by iodine containing drugs e.g. amiodarone (63). In the European Study 

Group of Hyperthyroidism a high proportion of iodine contamination was observed ranging 

from 18% in Graves disease to 54% in non-autoimmune hyperthyroidism (64). Severity of 

iodine deficiency, autonomous thyroid cell mass, the quantity of administered iodine and 

older age have been proposed as risk factors for the development of iodine induced 

hyperthyroidism (64). 

IV Clonal origin of thyroid nodules 

Studies that address the clonal expansion of a tumor have provided a valuable tool to decide 

the nature or etiology of a focal growth event being either neoplasia or hyperplasia. Whereas 

hyperplasia is a reversible outcome of an external trophic stimulus (e.g. iodine deficiency in 

the thyroid) neoplasia results from an intracellular defect (i.e. genetic alteration) and is 

irreversible (this definition of neoplasia in not equivalent with malignancy). For the thyroid 

gland a critical review of the early work on clonal analysis was given by Thomas et al. (65). 

Later, heterozygous polymorphisms in X-chromosome-linked markers (66) have been 

extensively used to demonstrate a predominant clonal origin of tumor tissues including the 

thyroid gland (67-71). Despite increasing technical concern (reviewed in (72)) clonal analysis 

is still a frequently used tool in tumor biology often seen as an intermediate step in pursuing 

11

the ultimate goal which is the detection of the molecular cause of neoplastic growth, namely 

mutations in the genomic DNA. Especially recent results of clonal analysis after PCR 

amplification of X-linked markers from micro-dissected tumors need to be interpreted with 

caution (73). The thyroid develops from a number of progenitor cells that migrate from the 

floor of the primitive pharynx called the median thyroid anlage (74). In females each 

progenitor shows a defined pattern of inactivation for most genes on one of the two X- 

chromosomes that is conferred to the progeny (75). Proliferation of these progenitors forms a 

cluster of cells (i.e. the thyroid patch) that share the same pattern of X-chromosome 

inactivation. If a sample for clonal analysis (e.g. from a micro-dissected tumor) lies entirely 

within such a patch/cluster an identical pattern of X-chromosome inactivation which implies 

monoclonality is not a reliable marker for neoplasia (72;76). Vice versa, without micro- 

dissection the distinction of monoclonal origin of samples from true neoplasia could be 

concealed by contamination with blood, connective, and, surrounding healthy tissue. In both 

cases a histochemical analysis of clonal origin that allows to examine the clonal architecture 

would be very helpful. However, available techniques can not be applied in general because 

the tissue under investigation has to meet several requirements (73;77). Nevertheless 

histology based clonal analysis (73;77) indicates a patch size in the thyroid gland that is much 

smaller than patch sizes determined with PCR using paraffin-embedded tissue sections (78). 

Moreover, in line with the histology based data our own study using the PCR approach on 

microdissected thyroid follicles demonstrates a polyclonal origin in about 25% of single 

follicles (Krohn, unpublished). If a hyperplastic lesion is likely to arise from a single patch 

then clonal analysis with the current methodology would have a strong bias toward showing 

monoclonality for this lesion. It is therefore crucial to consider the conditions that would 

allow to assume that a hyperplastic nodule could arise from a single thyroid patch. This 

decision mainly depends on the extend of thyroid patch size and the growth potential of a 

hyperplastic lesion. If the thyroid patch size is small a higher growth potential would be 

12

necessary to allow a hyperplastic lesion to develop from a few follicles into a macroscopically 

detectable thyroid nodule. Because data that would allow to determine this growth potential in 

vivo are not directly available we instead like to consider data that show the extend of thyroid 

hyperplasia after goitrogenic stimulation in animal models. These date suggest a rather low 

growth potential because thyroid enlargement under extrinsic goitrogenic stimulation (e.g. 

iodine deficiency or extended TSH stimulation) is rarely higher than 3- to 5-fold (4;79-81). In 

contrast, intrinsic or intracellular growth stimulation caused by genetic manipulation in 

transgenic mice leads in some cases to increases of thyroid mass in the range of 100-fold 

(10;82;83). If this difference also applies to focal stimulation, it is very unlikely that a 

hyperplastic thyroid lesion (caused by extrinsic stimuli) that originates only from a single 

patch would reach the cell mass of a normal thyroid nodule. Therefore, it is more likely that a 

macroscopically detectable thyroid hyperplastic nodule originates from more than one patch. 

If so, this nodule should be detectable as polyclonal, if a large part of the respective tissue is 

studied for X-chromosome inactivation. Studies of clonal analysis in our group therefore used 

DNA extracted from the entire nodular tissue. This approach very likely reduces the a strong 

bias toward showing monoclonality for a hyperplastic lesion. 

Our investigations of the clonal origin of autonomously functioning thyroid nodules and 

solitary cold thyroid nodules (both adenomas and adenomatous nodules) used a PCR 

approach to amplify the X-linked human androgen receptor from genomic DNA of female 

patients (84-86). After thorough screening for somatic mutations in these thyroid nodules (for 

details see Chapter V) we could demonstrate that thyroid nodules with a somatic mutation are 

predominantly of clonal origin (84-86). This is not surprising because it is in full agreement 

with the widely accepted paradigm in tumor biology that neoplasia (for definition see above) 

originates from a single mutated cell (87). Moreover, we were specially interested in data that 

would elucidate the etiology of mutation negative nodules. Interestingly, more than 50% of 

mutation negative cases from female patients show a monoclonal origin when tested for X- 

13

chromosome inactivation (84-86). This could indicate a neoplastic process with a mutation in 

a gene other than the TSHR, the Gsα protein or the ras family of oncogenes. Moreover, our 

finding of an overall frequency for the monoclonal origin of thyroid nodules at about 60-70% 

agrees with a number of other studies (67;68;70;71) and further underscores that thyroid 

nodules predominantly result from a neoplastic process with somatic mutations as the starting 

point (8). 

V Hot thyroid nodules 

A Signal transduction of HTN with and without TSHR mutations 

Both, growth and function of the thyroid are controlled by TSH (5). Although the activation 

of the TSHR preferentially leads to stimulation of the adenylyl cyclase via the Gsα-protein, at 

higher TSH concentrations an activation of the phospholipase C cascade by Gqα has also been 

shown (88;89). Moreover, there is evidence that the TSHR may be coupled to other members 

of the G protein family (88;90). However experimental data are frequently focused on the 

cAMP-branch of TSH signaling. Early work by Pisarev et al. demonstrated that cAMP 

elevation causes goiter (91). Moreover, in the thyroid gland and cultured thyroid epithelial 

cells as well as other endocrine tissues it is widely accepted that cAMP stimulates 

proliferation (92-95). More recently, transgenic models were studied to further understand 

TSHR signaling in more detail: Chronic in vivo stimulation of the cAMP cascade stimulates 

epithelial cell proliferation in vivo (82;96;97). A dominant negative cAMP response element 

binding protein blocks signalling downstream of cAMP and causes severe growth retardation 

and primary hypothyroidism (98). TSH/TSHR signaling generally controls iodine metabolism 

but only affects growth in the adult thyroid gland and not during embryonic development 

(99;100). 

Somatic point mutations that constitutively activate the TSHR were first identified by Parma 

and co-workers in hyperfunctioning thyroid adenomas (101). However, in different studies the 

14

prevalence of TSHR and Gsα mutations in autonomously functioning thyroid nodules has 

been reported to vary from 8 to 82% and 8 to 75%, respectively (101-112). These studies 

differ in the extent of mutation detection and the screening methods. A comparison with 

respect to the obvious differences between the studies has been done elsewhere (8;113;114). 

A comprehensive study of our group using the more sensitive denaturing gradient gel 

electrophoresis (115-117), revealed a frequency of 57% TSHR mutations and 3% Gsα 

mutations in 75 consecutive autonomously functioning thyroid nodules (86). These results 

raise the question of the molecular etiology of TSHR and Gsα mutation negative nodules. A 

possible answer is given by clonal analysis of these AFTNs which demonstrates a 

predominant clonal origin of thyroid nodules and implies a neoplastic process driven by 

genetic alteration (for details see IV). In a recent study (118) we found that AFTNs without a 

TSHR mutation show an increased expression of the tumor suppressor protein p53-binding 

protein 2, which interacts with p53 and specifically enhances p53-induced apoptosis but not 

cell cycle arrest (119). From this finding one could speculate that increased expression of this 

gene could increase apoptosis in AFTNs without a TSHR mutation and thus have a negative 

effect on the growth of the tumor. However, data on apoptosis in AFTNs do not allow a 

comparison with respect to the TSHR mutation status (120;121). Furthermore, the AFTNs 

without a TSHR mutation differ from the nodules harboring a TSHR mutation in their 

increased expression of two genes which are involved in the signal transduction of G protein 

coupled receptors: RGS 6 and GRK 2. Members of the RGS family have been shown to 

modulate the function of G proteins by activating the intrinsic GTPase activity of the alpha 

subunits (122), whereas G protein coupled receptor kinases play a role in the receptor 

desensitization (123). In general, a higher expression of these genes would rather restrict 

cAMP accumulation in AFTNs and could have an negative effect on functional autonomy. 

However, further experiments have to explore the importance of these genes in the etiology of 

AFTNs without TSHR mutations. 

15

AFTNs with TSHR mutations lack a clear genotype/ phenotype correlation (113). A similar 

finding is evident for germline TSH receptor mutations (124). Variable phenotypes associated 

with the same TSHR mutation could be the result of influences on signaling downstream of 

the TSHR. This modulation could have a number of targets like G protein coupling, receptor 

desensitization and internalization or cross-talk with other signaling cascades. Although our 

knowledge concerning these targets is far from complete recent findings are very promising. 

Firstly, the TSHR itself could be the subject of regulatory mechanisms that contribute to the 

etiology of AFTNs and the clinical phenotype. Voigt et al. (125) could show that ß-arrestins 

interact with the TSH receptor and are able to desensitize the receptor. Increased expression 

of beta-arrestin 2 in AFTNs could cause desensitization of the TSHR and thereby down 

regulate constitutive activation. A similar result could be caused by increased TSHR 

internalization due to increased expression of GRKs in AFTNs (118;126). Besides direct 

effects on the TSHR protein altered interaction with G proteins would be the next downstream 

level where modulation interferes with constitutive activation. For example the mutated 

TSHR could show a shift of the coupling specificity for G proteins (127). Such a shift could 

allow a more efficient activation of other downstream cascades (e.g. JAK/STAT pathway) 

through PKC in addition to cAMP and IP (128;129). Gene expression analysis in AFTNs 

with TSHR mutations in comparison to AFTNs without a TSHR mutation would support this 

hypothesis demonstrating an increased expression of JAK 1, protein kinase C beta 1 and zeta 

mRNA (118). Evidence for other cascades (e.g. RAS/RAF/MEK/ERK/MAP pathway) to play 

a role in constitutive TSHR signaling in AFTNs is currently missing. 

In addition to the intracellular signaling network that is connected to the TSHR, the 

extracellular action of different growth factors enhances the complexity of the signal flux into 

the thyroid cell. Growth factors like insulin-like growth factor I, epidermal growth factor, 

transforming growth factor β and fibroblast growth factor stimulate growth and 

dedifferentiation of thyroid epithelial cells (130;131). Studies, which have been focused on 

16

insulin and insulin-like growth factor, show a permissive effect of insulin and IGF-I on TSH 

signaling (132-136) and a cooperative interaction of TSH and insulin/IGF-I (137). Signal 

modulation of the TSHR that would define the etiology of AFTNs and the clinical phenotype 

could therefore take part at a number of stages and very likely involves genetic/epigenetic, 

sex-related, and or environmental factors. 

B Secondary/indirect effects of activating TSHR mutations 

Because constitutively activating TSHR mutations disturb the coordinated signal transduction 

network of the thyroid in a drastic way, subsequent changes in the signal transduction network 

can be expected. These alterations based on the constitutive activation of the TSHR signaling 

can be described as indirect or secondary effects of the activating TSHR mutations. The use 

of the microarray technique offers the advantage of a highly parallel analysis of gene 

expression to analyze changes between AFTNs and CTNs compared with their surrounding 

tissue. This approach also allows to evaluate which genes and groups of genes are most 

frequently affected in the molecular etiology of thyroid nodules and possibly deduce a 

molecular defect from the expression pattern. In a recent study using the Affymetrix 

GeneChip technology we could show a distinctly changed pattern of gene expression of the 

TGF-β signaling pathway between AFTNs with and without TSHR mutations and their 

normal surrounding tissue (figure 1 (118)). The type III TGF-β receptor, Smad 1, 3 and 4, as 

well as p300, a transcriptional co-activator, showed a decreased expression in AFTNs, 

whereas the inhibitory Smads 6 and 7 showed an increased expression in AFTNs. These 

findings suggest inactivation of TGF-ß signaling in AFTNs due constitutively activated 

TSHR (e.g. resulting from TSHR mutations). This assumption is supported by findings of 

Gärtner et al. (138), who could show a decreased expression of TGF-β 1 mRNA after TSH 

stimulation of thyrocytes. Because TGF-β 1 has been shown to inhibit iodine uptake, iodine 

organification and thyroglobulin expression (139;140), as well as cell proliferation in different 

17

cell culture systems (141-144), these and our novel findings suggest that inactivation of TGF- 

β signaling is a major prerequisite for increased proliferation in AFTNs (120;145). 

Eggo et al. (146) have shown that enhanced production of insulin-like growth factor binding 

proteins (IGFBPs) is correlated with inhibition of thyroid function, whereas the TSH-cAMP 

signaling is capable to inhibit IGFBP production. Moreover, recent studies (118;147) reveal a 

significantly decreased expression of insulin-like growth factor (IGF)-II and IGFBP 5 and 6 in 

AFTNs in comparison to their normal surrounding tissue. Taken together, the decreased 

expression of the IGFBPs, and of IGF-II in AFTNs are most likely secondary effects of the 

increased TSHR-cAMP signaling in AFTNs. 

VI Cold thyroid nodules 

With a frequency of about 85% cold thyroid nodules (CTNs) constitute the most abundant 

thyroid nodular lesion (for detailed definition and epidemiology see chapter I). The term 

“cold” indicates that this thyroid lesion shows reduced uptake on scintiscan. Because 

histologic diagnosis is typically employed to exclude thyroid cancer many investigations of 

thyroid nodules only refer to the histologic diagnosis of thyroid adenoma. This histologic 

entity should not be confounded with the scintigraphically characterized entity “cold nodule”, 

which like AFTNs or “warm nodules” (for the distinction see chapter I) can histologically 

appear as thyroid adenomas or adenomatous nodules according to the WHO classification (1). 

In this review we will focus on benign neoplastic lesions because substantial information 

concerning genetic events and molecular mechanism is available in the literature on human 

cancer in particular thyroid carcinoma that very likely also applies to benign neoplasia of 

thyroid follicular cells. In contrast, focal hyperplasia is not very well explained on the 

molecular level and has been discussed in detail elsewhere as the cause of thyroid tumors 

(148;149). As detailed in chapter IV, a monoclonal origin has been detected for the majority 

of thyroid nodules which implies nodular development from a single mutated thyroid cell. 

18

Hypotheses in studies that aim to understand the molecular or genetic causes of human 

cancer in general (150;151) or AFTN (8) and thyroid carcinomas (152) in detail have often 

also been applied to studies of cold thyroid nodules (e.g. ras mutations (153;154), for review 

see Wynford-Thomas (155)). In contrast to thyroid carcinomas where a number of genes have 

been implicated in the pathogenesis of these lesions (152;156) and in contrast to AFTN where 

constitutively activating TSHR mutations are very prevalent genetic events (7;8) knowledge 

concerning the molecular etiology of CTNs is limited. 

A Iodide transport and metabolism 

With reference to their functional status (i.e. reduced iodine uptake) failure in the iodide 

transport system or failure of the organic binding of iodide have been detected as functional 

aberrations of cold thyroid nodules long before the molecular components of the iodine 

metabolism were known (for review see Paschke and Neumann (157)). Later, a decreased 

expression of the Na + /I - symporter (NIS) in thyroid carcinoma and benign cold thyroid 

nodules suggested the molecular mechanism for the failure of the iodide transport (reviewed 

in (158-160)). Although the extent of decrease in NIS mRNA expression of cold thyroid 

nodules varies in different studies (160-163) reduction is in many cases very likely the result 

of hypermethylation in the NIS promoter (160). Moreover, in vitro studies suggest that 

reduced NIS mRNA expression could be caused by constitutive activation of RET or RAS 

genes (164-166). However, reduced NIS mRNA expression does not necessarily lead to 

reduced NIS protein expression (figure 2) (160). Furthermore and in contrast to other thyroid 

disorders with congenital iodide transport defects (for review, see (167;168)) no NIS gene 

mutation that would render this protein non-functional was detectable in CTNs (160). 

Therefore, the recently identified defective cell membrane targeting of the NIS protein is a 

more likely molecular mechanism that could account for the failure of the iodine uptake in 

CTNs (158;160;169). However the ultimate cause of this defect is currently unknown. 

19

Compared to iodine transport the organic binding of iodine is a multistep process with a 

number of protein components that still awaits final characterization (170). mRNA expression 

of enzymatic components (e.g thyroid peroxidase (TPO) or flavoproteins) and the substrate of 

iodination (i.e. thyroglobulin (TG)) have been quantified in CTNs without significant 

differences to normal follicular tissue (163;171). TPO, TG and thyroid specific oxidases 

(THOX) have been successfully screened for molecular defects especially in congenital 

hypothyroidism (172). Although, cold thyroid nodules could be considered as a form of focal 

hypothyroidism, somatic mutations in enzymes that catalyze organic binding of iodine would 

need to exert a growth advantage on the affected cell to cause the development of a thyroid 

nodule. At least in the case of inactivating mutations in the TPO or THOX genes growth 

advantage could result from a lack of enzyme activity which would not only reduce thyroid 

hormone synthesis but also follicular iodide trapping in organic iodocompounds. Because 

these compounds have been shown to inhibit thyroid epithelial cell proliferation 

(133;173;174) reduced synthesis could have a proliferative effect. Therefore, somatic TPO or 

THOX mutations could be a molecular cause of CTN. However mutations in the TPO gene 

could not be detected (175) and an ongoing screening for mutations in the THOX genes is 

also negative so far (Krohn, Paschke, Ris-Stalpers, unpublished). 

B Signaling proteins 

In addition to a failure in metabolic proteins that might explain the development of CTNs on 

the molecular level pathologic changes of signaling molecules might reprogram the growth 

stimulus and lead to clonal expansion of thyroid epithelial cells. Although not a particular 

subject of this review, much can be learned from findings in thyroid carcinoma. In this regard 

genetic changes (i.e. point mutations) that cause constitutive activation of the 

RAS/RAF/MEK/ERK/MAP pathway have been suggested as a key mechanism during tumor 

initiation or progression in thyroid follicular cells (for review, see (155)). So far, the only 

known molecular event that evidently causes such an activation in thyroid carcinomas and 

20

cold thyroid nodules is a mutation in one of the small RAS oncogenes (153). Recently BRAF 

mutations first detected in melanomas and with lower frequency in other cancers (176) have 

been detected in thyroid papillary carcinomas (177). They can also activate this pathway and 

might therefore also cause benign follicular lesions. Strikingly, both in colorectal and thyroid 

cancers BRAF mutations occur only in tumors that do not carry mutations in a RAS gene. In a 

recent study of 40 cold thyroid adenoma and adenomatous nodules we detected ras mutations 

in only a single case (85). Moreover, in the same set of CTNs we did not detect point 

mutations in the mutational hot spots of the BRAF gene (178). This is in line with the lack of 

BRAF mutations in benign follicular adenoma in other studies (177;179;180). So far only one 

study detected a single BRAF mutation in a set of 51 follicular adenoma (181). Instead of 

RAS and BRAF mutations there could be other molecular events that could constitutively 

activate the RAS/RAF/MEK/ERK/MAP pathway. Such candidate molecules include other 

members of the RAF gene family like RAF-1 or downstream genes like ERK or MAP 

kinases. However mutations in these genes have not been reported in benign thyroid lesion so 

far. Furthermore, molecular events that lead to activation of other cascades that exert synergy 

with MAP kinase signaling (e.g. cAMP signaling) or inactivation of independent cascades 

that restrict proliferation (e.g. TGF-ß signaling) could explain cold thyroid nodules. In 

addition to the TSHR, several G protein isoforms like Gi2alpha (182) or Gq, and G11 (183) as 

well as some candidate genes mediating downstream cAMP signaling like Epac and Rap1 

(184) have been screened in cold thyroid nodules for mutations. However, only a single 

somatic mutation in the Gi2alpha gene (182) was found in follicular adenomas. 

C Results of gene expression studies by arrays 

Currently, expression profiling of signaling proteins using microarray methodology is a 

promising approach which may contribute to further understanding the molecular events that 

lead to the development of AFTNs (147;185) or thyroid carcinoma (186-188). Functional 

21

characteristics of cold thyroid nodules suggest that mechanisms initiating growth but not 

leading to hyperfunction need to be defined. As far as future screenings for genetic defects are 

concerned expression profiling could describe the molecular mechanism and rule out a 

number of possible targets (e.g. because they are not expressed) or unmask alternative 

candidates. Moreover, as demonstrated for AFTNs, results of expression profiling might shift 

attention to other signaling cascades (for details on AFTNs see chapter V). For differentially 

expressed genes within these cascades a sequencing approach might then be warranted. In 

addition, knowledge of the molecular signature of CTNs and benign thyroid tumors in general 

could be very helpful to define differences between benign and malignant thyroid disease with 

diagnostic or therapeutic relevance. 

Recently, our group investigated 588 genes by cDNA expression arrays in three AFTNs and 

three CTNs as well as corresponding normal surrounding tissue. In general, changes in the 

expression of several signal transducing components were detected. Although this seems to 

reflect a disturbed signaling system, the results of that limited study did not allow to identify 

specific signal transduction cascades which might be involved in nodular development (147). 

To gain a higher resolution we compared gene expression for approximately 10,000 full- 

length genes between CTNs and their corresponding normal surrounding tissue (Eszlinger, 

Krohn and Paschke, submitted). Here regulation of gene expression in CTNs was most 

consistent for a group of several histone mRNAs. Increased expression of these histone 

mRNAs and of cell cycle associated genes like cyclin D1, cyclin H/cyclin dependent kinase 

(CDK) 7 and cyclin B most likely reflect a molecular setup for an increased proliferation in 

CTNs (189). In line with the low prevalence of ras mutations in CTNs (85), we find a reduced 

expression of ras-MAPK cascade associated genes which might suggests a minor importance 

of this signaling cascade. 

22

D Chromosomal aberrations 

Loss of heterozygocity (LOH), microsatellite instability and more recently gene 

rearrangements and chromosomal translocations as different forms of chromosomal aberration 

are considered important steps in carcinogenesis and have been investigated as potential 

markers to discern benign from malignant nodular disease. Findings of chromosomal 

aberrations and microsatellite instability in benign thyroid tumors although sometimes 

sporadic suggest that there is a difference in the extent of these DNA changes (190;191). 

Alternatively these results could stem from errors in histologic characterisation (192). 

Especially gene rearrangements unique to thyroid adenomas have recently been the focus 

(reviewed in (193)). These studies led to the identification of the thyroid adenoma associated 

gene (THADA) that encodes a death receptor interacting protein (194). 

Although also reported for thyroid follicular carcinoma (195) our finding of (LOH) at the 

TPO locus is characteristic for some CTNs (about 15%) but rather points to defects in a gene 

near TPO on the short arm of chromosome 2 (175). Moreover, after identification in a 

significant portion of follicular carcinomas (196) Pax-8/PPARγ gene rearrangement have also 

been reported for cold thyroid nodules (197) but seem to be a rare finding (198-200) or due to 

histologic misclassification of the thyroid nodules (192). Although the frequency of each of 

these DNA aberrations is rather low together these chromosomal changes need to be 

considered in the further elucidation of the molecular etiology of CTNs. 

VII Multinodular Goiter 

Multinodular goiter refers to an enlargement of the thyroid with deformation of the normal 

parenchymal structure by the presence of nodules. These nodules vary considerably in size, 

morphology and function (for detailed definition and epidemiology see chapter I). In areas 

without endemic goiter MNG is often referred to as sporadic non-toxic goiter. MNG usually 

develops in an already enlarged thyroid independent of the cause of hyperplasia (for review 

23

see (149). Over time (sometimes decades) many euthyroid multinodular goiters enlarge 

further, some develop subclinical hyperthyroidism and subsequently present as TMNG 

(4;201). The main epidemiologic determinants outlined in detail in chapter I for the 

development of MNG and TMNG are iodine deficiency (22), age, sex and duration of goiter 

in iodine deficient (4;17;18) and also in iodine sufficient areas (for review, see (202)). It is 

widely accepted that the basis for the development of nodular structures is an early stimulus 

that causes enlargement of the thyroid. However, clinical manifestations of MNG might only 

appear after a long period of time (sometimes up to 30 and more years). In general 

development of MNG proceeds in two phases: global activation of thyroid epithelial cell 

proliferation (e.g., as the result of iodine deficiency or other goitrogenic stimuli) leading to 

goiter and a focal increase of thyroid epithelial cell proliferation causing thyroid nodules. So 

far, the most common stimulus for local proliferation are somatic mutations (see chapter V 

and VI). 

A Mutagenesis as the cause of nodular transformation 

From animal models of hyperplasia caused by iodine depletion (79;203;204) we learn that 

besides an increase in functional activity a tremendous increase in thyroid cell number occurs. 

These two events very likely orchestrate a burst of mutation events. Although the enzymatic 

setup awaits further characterization (171) it is known that thyroid hormone synthesis goes 

along with increased H2O2 production and free radical formation (205), which may damage 

genomic DNA and cause mutations (206). As a consequence, the spontaneous mutation rate in 

the thyroid is almost 10-times higher than in other organs (e.g. compared to liver, Krohn 

unpublished). Together with a higher spontaneous mutation rate a higher replication rate will 

more often prevent mutation repair and increase the mutagenic load of the thyroid, thereby 

also randomly affecting genes crucial for thyrocyte physiology. Mutations, that confer a 

growth advantage (e.g. TSHR or Gsα protein mutations) very likely initiate focal growth. 

Hence autonomously functioning thyroid nodules are likely to develop from small cell clones, 

24

that contain advantageous mutations as shown for the TSHR in ‘hot’ microscopic regions of 

euthyroid goiters (9). 

B Etiology 

Epidemiologic studies, animal models and molecular/genetic data outline a general theory of 

nodular transformation. Based on the identification of somatic mutations and the predominant 

clonal origin of AFTNs we propose the following sequence of events that could lead to 

thyroid nodular transformation in three steps (figure 3). In the first step, iodine deficiency, 

nutritional goitrogens or autoimmunity cause diffuse thyroid hyperplasia. Then, at this stage 

of thyroid hyperplasia increased proliferation together with a possible DNA damage due to 

H2O2 action causes a higher mutational load with a higher number of cells bearing a mutation. 

Some of these spontaneous mutations confer constitutive activation of the cAMP cascade (e.g. 

TSH-R and Gsα mutations) that stimulate growth and function. Finally, in a proliferating 

thyroid growth factor expression (e.g. IGF-I, TGF-ß1 or EGF) is increased. As a result of 

growth factor co-stimulation all cells divide and form small clones. After increased growth 

factor expression ceases small clones with activating mutations will further proliferate if they 

can achieve self-stimulation. They could thus form small foci, which will develop into thyroid 

nodules. This mechanism could explain AFTNs by advantageous mutations that both initiate 

growth and function of the affected thyroid cells as well as CTNs by mutations that stimulate 

proliferation only (e.g. ras mutations or other mutations in the RAS/RAF/MEK/ERK/MAP 

cascade). Moreover, nodular transformation of thyroid tissue due to TSH secreting pituitary 

adenomas (207), nodular transformation of thyroid tissue in Graves’ disease (208) and in 

goiters of patients with acromegaly (209) could follow a similar mechanism because thyroid 

pathology in these patients is characterized by early thyroid hyperplasia. 

As an alternative to the increase of cell mass and as illustrated by those individuals who do 

not develop a goiter when exposed to iodine deficiency the thyroid might also adapt to iodine 

deficiency without extended hyperplasia (210). Although the mechanism that allows this 

25

adaptation is poorly understood preliminary data from a mouse model suggest an increase of 

mRNA expression of TSH-R, NIS and TPO in response to iodine deficiency which might be a 

sign for increased iodine turnover in the thyroid cell in iodine deficiency (Krohn 

unpublished). 

VIII Pathogenesis and genetic etiology of euthyroid goiter 

Considering recent advances in the methodology of genetic analysis the genetic etiology of 

goiter is under-investigated. This applies especially to studies that target the genetic basis for 

euthyroid and toxic MNG. As outlined in the preceding chapter the development of nodular 

goiter is very likely a continuous process that starts with thyroid hyperplasia and simple 

goiter. Therefore, defects in genes that play an important role in thyroid physiology and 

hormone synthesis (see chapter ‘Candidate loci’) could be genetic factors that predispose for 

the mechanisms that lead to multinodular goiter. Such defects likely lead to 

dyshormonogenesis as an immediate response and might not directly explain nodular 

transformation of the thyroid. In this chapter we therefore also consider genetic studies that 

concern other forms of goiter. 

A Family and twin studies 

Although lack of iodine is the most prevalent factor for simple goiter as well as endemic 

goiter, other causes are likely. Familial clustering of goiters and the female predominance of 

goiters are the two major arguments suggesting a genetic background for euthyroid goiters. 

Family and twin pair studies in endemic and non endemic areas clearly demonstrated a 

genetic predisposition for goiter development. Within a Greek region endemic goiter affects 

some families more than others (211). This could provide evidence for a genetic etiology, 

though environmental factors that differ between families must also be considered (212). The 

familial aggregation of goiters in Greece was confirmed in a subsequent study (213). The 

26

progeny of affected persons were more often affected by goiter than the descendants of 

unaffected subjects. Likewise, other euthyroid goiter family studies in Greece, Slovakia and 

Africa also lead to the conclusion that a genetic predisposition is present in the affected 

individuals (211;214;215). In addition, more rapidly growing goiters in a subgroup of school 

children (15-20%) in spite of iodine supplementation (214) and differences in thyroid volume 

of adolescent siblings with sufficient iodine intake in Slovakia (216) also supports a genetic 

influence on thyroid growth. Moreover, studies in Greek populations have shown the 

persistence of endemic goiters in certain regions despite iodine supplementation (217). 

Familial occurrence of euthyroid goiter in an iodine replete region in Sweden was reported for 

41% of the patients with goiter with an even higher frequency of familial occurrence in those 

individuals with prepubertal development of the goiter (218). Even though, family studies are 

a reliable method to determine goiters in many family members over several generations, it is 

impossible to definitively conclude whether the members shared the same susceptible genetic 

make up or the same environment. Therefore, twin studies are more informative in 

demonstrating a genetic component in the etiology of goiter. Hence, several investigations 

have provided evidence that there is a predisposing genetic background for goiter in twins. In 

endemic as well as nonendemic areas female monozygotic twins (80% (213;219) and 42% 

(220), respectively) have a higher concordance rate for goiter than female dizygotic twins (40- 

50%, (213;219) and 13%, (220). Twins of the same sex are supposed to share the same family 

environment. Therefore, the increased concordance was attributed to greater genetic similarity 

characterizing the monozygotic twins. Contribution of genetic susceptibility to the 

development of goiter was calculated to be 39% in endemic regions (219). Moreover, a study 

of 5479 monozygotic and dizygotic twins (220) performed by path analyses (structural 

equation modelling) suggests that the genetic predisposition to develop goiter is 82 % with 18 

% according to individual environmental factors in a nonendemic area. However, the 

27

previously reported twin studies show the importance of both hereditary and environmental 

factors (Hegedus, Bonnema, & Bennedbaek 2003). 

B Candidate loci 

Because of their important role in thyroid physiology and hormone synthesis, thyroglobulin 

(TG) and thyroid peroxidase (TPO), the sodium–iodide–symporter (NIS), pendrin gene (PDS) 

and the TSHR are major candidate genes for familial euthyroid goiters. 

Studies of hypothyroid goiters have identified several genetic defects in TG and TPO (172). 

Congenital goiter and hypothyroidism caused by qualitative and quantitative defects of the 

TG gene were described by Medeiros-Neto (221). Other studies have also shown a link 

between the TG gene and congenital goiter and hypothyroidism (222-229). Furthermore, an 

inherited abnormality in TG synthesis leading to a lower content of TG in the thyroid gland 

was reported by Yoshida et al. (230) and a single amino acid substitution in the TG protein 

(Leu2366Pro) causes endoplasmic reticulum storage disease as determined in the cog/cog 

mouse (231). Although TG was postulated to be a major candidate gene for euthyroid simple 

goiter only one genetic variation associated with euthyroid goiter has been identified in the 

TG gene up to date. Corral et al. (232) found a G - to T substitution at position 2610 of the 

TG cDNA. This resulted in replacement of histidine for glutamine at codon 870. This 

sequence alteration was located within exon 10 of the TG gene and was present in 25 of 26 

members of 3 families affected by euthyroid goiter. However, Perez–Centeno et al. (233) 

found the same point mutation in thyroglobulin exon 10 gene only in one of 36 patients with 

endemic euthyroid goiter. Hishinuma et al. (226;229) found two novel cystein substitutions in 

thyroglobulin, which caused defects in the intracellular transport of thyroglobulin in patients 

with a variant type of adenomatous euthyroid goiter. Gonzalez–Sarmiento et al. (234) 

identified a large heterozygous deletion within the TG gene in a study of 50 cases affected 

with nonendemic goiter. The deletion involved the promotor region and the exons 1 to 11 of 

the TG gene and was associated with euthyroid goiter. 

28

Mutations responsible for dyshormogenesis have also been described in the TPO gene. TPO 

catalyzes the oxidation of iodide to an iodination species that forms iodothyrosines and 

iodothyronines. Defects of TPO synthesis caused by a heterogenous spectrum of TPO 

mutations (235-240) have been reported to result in reduced TPO activity in combination with 

total iodide organification defect (TIOD). Hagen et al. (241) described an intelligent, 

euthyroid child with goiter. Together with her affected sister she showed no iodide 

peroxidation or thyrosine iodination activity. Likewise, a euthyroid woman with a recurrent 

goiter and partial iodide discharge was described by Pommier et al. (242). She had normal 

iodide peroxidation but deficient thyroglobulin iodination. However, these are the only two 

examples for TPO mutation resulting in euthyroid goiters. Most of the previously reported 

homozygous or compound heterozygous mutations in the TPO gene lead to goiter and 

hypothyroidism (236;243;244). 

Since the cloning and molecular characterization of the human NIS gene (245) several defects 

in this gene have been detected in patients with different phenotypes of thyroid diseases (246). 

A heterozygous T354P mutation results in congenital hypothyroidism and goiter (247-251). 

However, two studies reported that the homozygous T354P mutation is associated with 

euthyroidism and goiter (252;253). Moreover, other allelic variants producing deletion, 

missense or truncation of the NIS protein have been described (254;255). 

Mutations in the PDS gene cause Pendred syndrome characterized by congenital sensorineural 

hearing loss combined with goiter. In Pendred syndrome the thyroid enlargement typically 

begins in childhood and can vary between and within families (256;257). Reported goiter 

sizes vary from small nodules to large multinodular goiters (258). Almost all affected 

individuals are clinically and biochemically euthyroid. Positive perchlorate tests suggest that 

the PDS defects impair the organification of iodide. Different PDS mutations, each 

segregating with the disease in the families in which they occurred, have been identified (259- 

263)(85). Most of them are loss-of-function mutations which directly cause thyroid disease in 

29

Pendred syndrome. Therefore, the PDS gene is a candidate gene for development of euthyroid 

goiter. 

Furthermore, the TSHR on chromsome 14q31 could be a candidate gene for euthyroid goiter 

according to its central role for thyroid function and growth. TSHR germline mutations have 

been found in rare cases of euthyroid familial goiter (7) (see also chapter IA). Moreover, a 

germline genetic variation in codon 727 of the TSHR gene (Asp ->Glu (264)) has been 

associated with toxic multinodular goiter in iodine deficient areas (265). However, the wild 

type TSHR response to TSH was very low in this study and this in vitro finding was not 

confirmed in a further study (266;267). Moreover, the putative predisposition for toxic 

multinodular goiter was based on functional analysis of the TSHR codon 727 variation which 

revealed a higher cAMP response compared to the wild type receptor. However, this in vitro 

finding was not confirmed in a recent study (266). Recently Peeters et al. (268) reported that 

the homozygous C variant (coding for aspartic acid) of the D727E polymorphism was 

associated with a lower serum TSH level in 156 healthy blood donors. Although this finding 

supports a possible functional relevance of this polymorphism the role of a lower TSH level in 

the development of MNG is unknown. 

C Linkage analysis 

Over the last years linkage analyses became a reliable method to identify novel susceptibility 

loci for both mendelian and complex diseases in large families or affected sib pairs by using 

genetic markers for microsatellite DNA or repetitive sequences in the entire human genome. 

Several different susceptible areas have been discovered in large families with non-mendelian 

transmission of euthyroid familial goiter. The multinodular-goiter-1 locus MNG-1 on 

chromosome 14q31 was first reported by Bignell (269) as the result of a genome wide linkage 

analysis of a large Canadian family with 18 patients affected with multinodular goiter. A 

maximum two point LOD score of 3.8 at D14S1030 and a multipoint LOD score of 4.88, 

defined by D14S1062 and D14S267 was calculated. In a further study a family with recurrent 

30

euthyroid goiters was investigated for linkage to the same candidate region (270). According 

to a dominant pattern of inheritance with full penetrance, indication for linkage was obtained 

by a maximum two point LOD score of 1.5 at marker D14S1030 at the MNG-1 locus. 

Moreover, a maximum multipoint LOD score of 1.49 was obtained for the region between the 

TSHR and the MNG-1 candidate loci. The haplotype cosegregation of microsatellite markers 

confirmed the entire chromosomal segment between both loci on chromosome 14q31 as a 

positional candidate region for nontoxic goiter. Although the TSHR was a first line candidate 

gene sequence analysis of the TSHR only revealed several previously reported 

polymorphisms. In the study by Bignell et al. the TSHR was previously clearly excluded as a 

candidate gene (269). Another study reported the analysis of an Italian - three generation 

pedigree, including 10 affected females and 2 affected males (271). An X-linked dominant 

pattern of inheritance was observed. The investigation of 18 markers spaced at 10 cM 

intervals on the X-chromosome revealed evidence for linkage at marker DXS1226 with a 

significant LOD score of 4.73. These findings led to the conclusion that defects in the Xp22 

region caused thyroid disease in this family. Moreover, the haplotype inspection reduced the 

critical interval to 9.6 cM between the markers DXS1052 and DXS8039. 

In view of the different susceptibility loci for euthyroid goiter a heterogenous mode of 

inheritance for euthyroid goiter is very likely. Linkage analysis for the thyroid candidate 

genes has been performed in four German and one Slovakian family (272). The candidate 

genes were also analyzed assuming different recombination fractions for the microsatellite 

markers in two point and multipoint analysis. Linkage analysis results of this study were not 

significant enough to definitely exclude or confirm linkage to the investigated candidate genes 

TG, TPO and NIS. To date, there is no evidence for or against susceptibility of the 

investigated candidate genes for euthyroid familial goiter. Since linkage to MNG-1 (14q31) 

was previously reported in two families (269;270) and Xp22 in a single family (271), the four 

German families and one Slovakian family were also investigated to test a more general 

31

validity of these candidate regions (272). However, the absence of a correlation of inheritance 

patterns for the investigated markers in the families and the nonsignificant LOD scores 

determined according to the Lander-Kruglyak guide suggested a lower probability for MNG-1 

and Xp22 as major monogenic causes for the etiology of euthyroid goiter. Moreover, in two 

families a very weak indication for linkage to PDS and Xp22, respectively, was identified 

(272). Furthermore, the nonsignificant LOD scores calculated in this study suggest, that the 

strongest genetic locus detectable by linkage is unknown to date and that it is probable that 

different candidate genes or loci cause euthyroid goiter in different families. In conclusion, 

these studies gave further indications for genetic heterogeneity of euthyroid familial goiters. 

To discover novel and more general candidate regions or genes we performed a genome wide 

scan to detect susceptibility loci that predispose for euthyroid goiter using 450 microsatellite 

markers in 18 Danish, German and one Slovakian family, comprising 79 affected and 68 

unaffected family members (273). Assuming genetic heterogeneity and a dominant pattern of 

inheritance four novel candidate loci on chromosomes 2q, 3p, 7q and 8p were identified. Four 

families showed linkage to the 3p locus whereas the loci 2q, 7q and 8p each showed linkage 

in one family. The haplotype inspection delimited a critical interval of 16 cM on 3p (figure 4). 

Within this interval the thyroid hormone receptor beta (THRB) is mapped. Our mutation 

screen also included the two thyroid hormone receptor interactor genes 6 and 12 on 7q and 2q 

in addition to the THRB gene (273). However, sequencing of all these candidate genes 

revealed no germline mutations, that would co-segregate with the goiter in the affected 

families. In conclusion, these genetic studies confirm that genetic heterogeneity is likely to 

explain the identification of different candidate loci like MNG-1 (269;270) and Xp22 (271) 

in several families. 

Most cases of familial goiter present an autosomal dominant pattern of inheritance. However 

for the majority of euthyroid goiter cases a multifactorial genesis with complex interactions of 

environmental factors like iodine deficiency, cigarette smoking, age, sex and certain drugs or 

32

emotional stress on a genetic background is more likely. Loci from linkage analysis or 

association studies could provide important genetic risk factors in a more complex genetic 

background. 

A Therapeutic implications 

IX Perspectives 

It is still poorly understood, how genes interact with different environmental factors (33). 

Therefore we have actually no ideal treatment for euthyroid benign (multi-)nodular goiter. 

Most clinical trials (56;57;274-281) investigated the efficacy of levothyroxine to suppress 

TSH and to arrest further growth or reduce the size of thyroid nodules. Thyroxine suppressive 

therapy is given with the hope that nodules might decrease in size. However, these studies 

reported contradictory results concerning the investigated endpoint (i.e. reducing the size of 

thyroid nodules). Moreover, the benefit of arresting growth or reducing the size of a thyroid 

nodule has not been conclusively answered because there are controversial reports on a 

possible correlation between thyroid nodule size and the development of thyroid epithelial 

cell carcinomas (52;282-284). 

Moreover, a decrease in serum TSH is related to increasing nodularity and size of the thyroid 

(4). Cross-section studies provide no evidence that the stimulation of thyroid growth or 

thyroid function through serum TSH is responsible for thyroid nodule growth (285;286) 

because patients with benign, cold thyroid nodules did not exhibit elevated TSH levels in 

comparison to controls (56;57;274;277-279). 

Furthermore, TSH suppression may lead to hyperthyroidism, reduced bone density and atrial 

fibrillation (287;288) and levothyroxine therapy can lower the intrathyroidal iodine content 

(289-291). The pathophysiological rationale for levothyroxine therapy of thyroid nodules with 

the aim of reducing their volume is therefore questionable. 

33

Moreover there is an uncertainty about predictors of response like clonality (8;84), growth 

(52), size at time of diagnosis (282-284), or cell-rich nodules (292)). 

Since thyroid nodules, thyroid autonomy and thyroid cancer were more often detected in 

iodine deficiency areas than in iodine sufficient areas (2;18;33;293), area wide iodine 

supplementation became the first choice in thyroid nodule prevention (24). Although iodine 

supplementation is an adequate therapy for nodular goiter (291;294) this option is often 

ignored. Possible benefits of treating or preventing thyroid nodule (growth) could be the 

avoidance of thyroid nodules/goiter associated symptoms (hoarseness, pain, hyperthyroidism, 

hypothyroidism), more rarely prevention of thyroid malignancy, prevention of surgical 

intervention and it’s related risks (295). In addition, this could lead to reduction of costs for 

common surgical interventions and postoperative pharmacotherapy (296). Therefore, the 

benefit of treating or preventing thyroid nodules is more likely prevention of clinical disease 

rather than reduction of nonclinical disease (295). In the authors’ view future studies should 

include patient-relevant outcomes like thyroid cancer incidence, health-related quality of life 

and costs. Multicentre studies are needed to investigate whether thyroid nodule growth is 

associated with an increased frequency of thyroid malignancies. 

B Diagnostic implications 

Evaluation of patients with nodular thyroid disease is directed at two aspects: exclusion of 

thyroid malignancy and definition of the functional and if possible pathomorphologic 

character of the nodule to stratify the best treatment approach. 

Diagnosis of thyroid malignancy is ultimately based on the histological examination but can 

be strongly suggested clinically e.g. by the presence of a rapid growing nodule, cervical 

lymph nodes, sudden onset of hoarseness and almost established on the basis of a malignant 

FNAC of the thyroid nodule (38;39;41). However, despite this clear-cut approach, the 

34

ultimate challenge in past and present thyroidology remains the identification of generally 

very rare thyroid cancer amongst the highly prevalent condition of nodular thyroid disease. 

This is not only reason for concern of the affected patients, and a daily task for all doctors 

dealing with thyroid disease, but increasingly poses an economic problem in times of limited 

health care budgets. 

Hence many studies and reviews have been dedictated to the resolution of this problem: There 

is agreement that both ultrasonography and thyroid scintiscan add little to nothing to the 

clarification of the benign or malignant nature of a nodule, with the exception that “hot” i.e. 

autonomously functioning thyroid nodules very rarely represent malignancy (38;39;41). 

Furthermore, it is widely acknowledged that fine needle aspiration cytology represents the 

most sensitive and specific means for pre-operative diagnosis of thyroid malignancy (40;297). 

The drawback remains that FNAC is only reliable if performed and analysed by a thyroid 

expert team (40;41). Besides it will be impossible to perform FNAC in all patients with 

nodular thyroid disease, which in countries with iodine deficiency may affect up to 30% and 

more of the adult population (18). Guidelines have therefore defined a nodule size of at least 

10-15 mm and/or hypofunctionality as indications for FNAC (38;39;41). However, it is 

unclear how to proceed in case of the much more frequent multinodular goiters, which 

probably harbor the same malignancy risk as solitary lesions (2), and a pragmatic approach 

here may be to perform FNAC of the prominent “cold” nodule (33) or to recommend thyroid 

surgery in cases of diagnostic uncertainty. The same pragmatic but not evidence guided 

approach is to perform surgery in patients with risk constellations e.g. past history of 

radiation, family history of thyroid cancer etc. 

Furthermore, even in an ideal setting e.g. in a specialist thyroid clinic, FNAC may be “non- 

diagnostic” or “suspicious” in more than 20% of cases. What are the options then? If FNAC 

has been non-diagnostic it needs to be repeated (298-300). “A number of at least six clusters 

35

of thyrocytes on each of at least two slides prepared from separate aspirates" has been 

proposed by Hamburger as a quality criterion for a diagnostic thyroid FNAC (301). However, 

recently Oertel has critically discussed that adequacy of the specimen cannot be based solely 

on the cell count (292). In case of ”suspicious” FNACs, which represent 10-20% of all 

FNACs (41) markers could be helpful, in particular to discriminate follicular adenoma from 

follicular carcinoma from follicular variant of papillary carcinoma and to distinguish Huerthle 

cell adenoma and cancer etc. (302). However, since much but still too little is known about 

the molecular etiology of the “common” thyroid nodule, it is even more difficult to define a 

marker of benignity versus malignancy although many candidates have been screened and 

proposed, some of which indeed seem promising e.g. Galectin-3, thyroperoxidase (MoAb47), 

PAX-8/PPARγ rearrangements, BRAF mutation (152;303-305), but none of which have 

made their way into routine diagnostics yet. In contrast novel technologies in particular 

microarray and proteomics methodolgies will almost certainly contribute to change this rather 

pessimistic state-of-the-art situation. Through these methods, which are increasingly applied 

in research labs everywhere, we have the possibility for the first time, to perform genome and 

proteome wide screening studies and despite all drawbacks they may in fact be ideally suited 

to identify useful markers similary to the in vivo situation, they are also performed as “cross 

sectional” studies. Contribution will also come from the likewise many genome wide linkage 

studies, which contributed to identify a number of disease “genes” in the past (306). One 

example is familial euthyroid goiter, discussed in chapter VIII. 

Which diagnostic markers would then be needed besides the important cytological 

differentiation of the above discussed entities of thyroid pathology? 

It would be ideal to have markers which indicate which nodules, on the long term, may turn 

into thyroid malignancy: More realistically, it could be feasible to define markers which 

correlate with augmented nodule growth or which correlate with ongoing thyroid de- 

differentiation. Clarification of the molecular characteristics and application of markers which 

36

define increased nodule growth and de-differention will allow formation of nodule subgroups. 

This would be the basis for a therapeutic strategy study to determine whether e.g. iodide 

supplementation, combination of L-T4 and iodine, no therapy, ethanol injection, radioiodine 

or surgery are indicated for which nodule subgroup. 

37

Figure legends 

Figure 1 

Differential mRNA expression in components of TGF β signaling in AFTNs (118) 

Gene expression analysis revealed a significantly decreased expression (green colored boxes) 

of the type III TGF-β receptor, Smad 1, 3 and 4 and the E1A-associated protein p300 in 

AFTNs in comparison to their normal surrounding tissue, whereas the inhibitory Smad 6 and 

7 are characterized by a significantly increased expression (red colored boxes). These findings 

argue for an inactivation of the TGF-β signaling cascade in AFTNs, which is normally 

responsible for inhibition of iodine uptake, iodine organification, thyroglobulin expression 

and cell proliferation in thyroid cells. 

Figure 2 

Immunohistochemical analysis of NIS expression in thyroid nodules and surrounding 

tissues (307) 

A: CTN 65 - nodule tissue; predominant intracytoplasmic NIS staining, only partly cell 

membrane immunoreactivity, B: CTN 65 - surrounding tissue; hNIS protein immunoreactivity 

is very low and heterogeneous, C: CTN 13 - nodule tissue, mostly 

intracytoplasmic immunoreactivity, D: toxic thyroid nodule; NIS cell membrane 

immunoreactivity is predominant 

Figure 3 

Hypothesis for thyroid nodular transformation. The starting point for the development of 

MNG is hyperplasia induced by goitrogenic stimuli (e.g. iodine deficiency). Iodine deficiency 

increases mutagenesis directly (production of H2O2/free radicals) or indirectly (proliferation 

and increased number of cell divisions). Subsequently, hyperplasia forms cell clones. Some of 

38

them contain somatic mutations of the TSH-R leading to autonomously functioning thyroid 

nodules (red spots) or contain mutations that lead to dedifferentiation and therefore cold 

thyroid nodules or cold adenoma (blue dots). 

Figure 4 

The figure shows the mapping strategy for susceptible chromosomal regions responsible for 

euthyroid goiter at chromosome 3p. a) highly polymorphic microsatellite markers on 3p, used 

to decide linkage of euthyroid goiter to this locus. b) shows the location of the peak NPL 

score which indicates linkage of euthyroid goiter. 

39

Table 1: Natural course of benign thyroid nodules in studies in iodine deficient and iodine sufficient areas with more than 2yr. follow-up. 

study country number of 

patients 

Papini et al. 

(56) 

Brander et 

al. (50) 

Alexander et 

al. (51) 

Kuma et 

al.(53)) 

Quadbeck et 

al. (55) 

Knudsen et 

al. (3) 

Italy 41 multicentre 

study 

location assessment follow-up definition of 

growth 

10MHz 

ultrasound 

inland 34 Cohort study 7.5MHz 

ultrasound 

USA 268 thyroid 

nodule clinic 

5-15MHz 

ultrasound 

Japan 134 Hospital Palpation 

and 7.5MHz 

ultrasound 

Germany 109 Endocrine 

Clinic 

7.5MHz 

ultrasound 

Denmark 45 Cohort study 7.5MHz 

ultrasound 

5 yr. > 12% increase in 

volume 

growth comment 

56% placebo group in L-T4 

study 

4.9-5.6 yr. not defined 35% investigation also of 

lesions < 10mm 

1 month to 5 

yr. 

> 15% increase in 

volume 

39% growth predominantly in 

solid nodules 

9-11 yr. not defined 21% > 40% (80% of cystic 

nodules) of nodules 

decreased or disappeared 

3-12 yr. > 30% increase in 

volume 

2 yr. > 5mm change in 

diameter 

50% no correlation with nodule 

function, age and gender 

2% 10% decreased only 

„cold“ nodules studied

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64

ligand 

TGF β 

TGF β 

type III 

TGFβR 

type I/II 

TGFβR 

receptors 

Smad 1 

Smad 2 

receptor 

Smads 

Smad 3 

Smad 5 

Smad 4 

coregulatory 

Smads 

inhibitory- 

Smads 

Smad 7 

Smad 6 

P300 

inhibition of 

thyroid cell 

differentiation and 

cell proliferation

impaired 

hormone 

Synthesis 

H2O2 ? 

free radicals ? 

hypertrophy 

hyperplasia 

proliferation 

mutagenesis 

single cells with 

somatic mutations 

adaptation by more 

efficient iodine 

clearence, trapping, 

metabolism 

and increased gene 

expression 

goiter with cell 

clones containing a 

somatic mutation 

. 

. 

. 

. 

. 

. 

. 

. 

expansion of cell 

clones 

with advantageous 

mutations leading to 

hot (or cold) nodules

a) 

D3S4545 

D3S1259 

D3S3038 

D3S1266 

D3S2432 

D3S1768 

D3S2409 

b) 

2.0 

1.0 

0 

NPL score 

1.0 

2.0 

3.0 

16 cM interval

Molecular pathogenesis of euthyroid and toxic multinodular goiter

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