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Original Studies |
Protein Gene in 31 Toxic Thyroid Nodules1
Department of Internal Medicine III, University of Leipzig, Leipzig, Germany
Address all correspondence and requests for reprints to: Prof. Dr. R. Paschke, University of Leipzig, Third Medical Department, Philipp-Rosenthal-Straße 27, D-04103 Leipzig, Germany.
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Abstract |
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protein (gsp) mutations in toxic
thyroid nodules (TTNs) reported conflicting results, most likely also
related to the different screening methods applied and the
investigation of only part of exon 10 of the TSHR. Therefore, we
screened a consecutive series of 31 TTNs for both TSHR and
gsp mutations by direct sequencing of exon 9 and the
entire exon 10 of the TSHR gene and exons 7–10 of the
gsp gene. Somatic TSHR mutations were identified in 15
of 31 TTNs. TSHR mutations were localized in the third intracellular
loop (Asp619Gly and Ala623Val), the sixth
transmembrane segment (Phe631Leu and Thr632Ile,
Asp633Glu) and the second extracellular loop
(Ile568Thr). One mutation was found in the extracellular
TSHR domain (Ser281Asn). Two new TSHR mutations were
identified. One involves codon 656 in the third extracellular loop
(Val656Phe). The other new mutation is a 27-bp deletion in
the third intracellular loop resulting in deletion of 9 amino acids at
codons 613–621. Transient expression of the new TSHR mutations in
COS-7 cells demonstrated their constitutive activity. No mutation was
found in exons 7–10 of the gsp gene. This finding was
confirmed by an allele-specific PCR for mutations in gsp
codons 201 (Arg
His, Cys) and 227 (GlnHis, Arg). Our data indicate
that constitutively activating TSHR mutations can be found in 48% of
TTNs and thus currently represent the most frequent molecular mechanism
known in the etiopathogenesis of TTNs. Moreover, the absence of
gsp mutations in our series argues for an only minor
role of these mutations in TTNs. Constitutive activation of the TSHR by
a deletion in a region that might be involved in G protein coupling of
the TSHR offers new insights into TSHR activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein
(gsp) genes have previously been identified in toxic thyroid
nodules (TTNs) (1, 2). Furthermore, TSHR germline mutations with
similar properties have been identified in families with autosomal
dominant nonautoimmune hyperthyroidism (3, 4, 5, 6) and in four cases of
sporadic congenital nonautoimmune hyperthyroidism (7, 8, 9, 10). As chronic
overstimulation of the cAMP pathway in thyrocytes by ectopic expression
of the adenosine A2 receptor in thyroid tissue of transgenic mice has
been shown to result in thyroid hyperplasia as well as hyperthyroidism
(11), constitutively activating TSHR and gsp mutations could
provide a coherent explanation for the clinical findings of autonomous
growth and hyperfunction in toxic nodules and for toxic hyperplasia in
the case of germline mutations.
The frequency of activating TSHR mutations in TTNs was first reported
as 82% (12). However, consecutive studies found a prevalence of
8–75% (13, 14, 15, 16, 17). Likewise, the reported gsp mutation
frequency in TTNs ranges from 4–38% (2, 15, 17, 18). These highly
variable prevalences of TSHR and gsp mutations in TTNs were
reason to speculate on additional mutational events or genetic and
epigenetic factors influencing the TSHR and gsp mutation
rate in TTNs. However, because of major methodological differences,
only the results of few studies can be compared to each other (Table 1
). Firstly, most studies have screened
parts of the TSHR transmembrane domain (1, 13, 14, 15, 19), yet
constitutively activating mutations in the TSHR (20, 21) as well as
other G protein-coupled receptors for which constitutively activating
mutations have been reported (22, 23) are spread throughout the
transmembrane domain, which in the case of the TSHR is encoded by exon
10. Moreover, TSHR mutations in exon 9 encoding part of the
extracellular TSHR domain have only very recently been reported (17).
Secondly, different methods have been used for detection of TSHR
mutations. Analysis by restriction enzymes (14) will only identify
known mutations; single strand confirmation polymorphism (19) is known
to be less sensitive (24) than direct sequencing (12, 13, 14, 15, 16, 17). Thirdly, DNA
extracted from paraffin-embedded tissue (18, 19) yields mostly highly
fragmented DNA, which is more difficult to amplify for mutation
detection than DNA extracted from frozen tissue (12, 13, 14, 15, 16). Fourthly,
mutation screening in TTNs was mostly performed either for the TSHR
gene (1, 12, 13, 14, 19) or the gsp gene (2, 18) rather than for
both genes in the same series (15, 16, 17). Fifthly, it is unclear whether
previous studies investigated unselected consecutive TTNs of patients
undergoing thyroid resection that would be necessary for conclusions on
mutation prevalence in TTNs or selected cases (25, 26).
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For these reasons and to minimize bias through sample selection, we screened a consecutive series of 31 patients with TTNs for both TSHR and gsp mutations by direct sequencing of exon 9 and the entire exon 10 of the TSHR and exons 7–10 of the gsp gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specimens of 31 consecutive TTNs and adjacent normal thyroid tissue of patients undergoing thyroid resection for treatment of their hyperthyroidism were obtained at surgery. Tissue samples were shock frozen in liquid nitrogen. The diagnosis of TTN was based on the clinical finding of hyperthyroidism with suppressed TSH levels and raised free T3 (FT3) and/or free T4 (FT4) values, negative screening for thyroid antibodies, and increased circumscribed technetium uptake by the nodule with suppression of surrounding thyroid tissue on scintiscan. Extraction of genomic DNA was performed from the hyperfunctioning nodules and surrounding normal thyroid tissue with a Qiagen Tissue Kit (Qiagen, Chatsworth, CA).
The study was approved by the local ethics committee. Informed consent was obtained from all patients before surgery.
PCR and sequencing
TSHR. Two overlapping fragments encompassing the entire exon 10 were amplified by PCR. The primers for the N-terminal fragment (868 bp) were: forward primer, 5'-TGG CAC TGA CTC TTT TCT GT-3'; and reverse primer, 5'-GTC CAT GGG CAG GCA GAT AC-3'. The primers for the C-terminal fragment (875 bp) were: forward primer, 5'-ACT GTC TTT GCA AGC GAG TT-3'; and reverse primer, 5'-GTG TCA TGG GAT TGG AAT GC-3'. For amplification of exon 9 of the TSHR, the primers were: forward primer, 5'-TCA TCT CCC AAT TAA CCT CAG G-3'; and reverse primer, 5'-GCT TCC AAT TTC CTC TCC AC-3' (27).
gsp.
For amplification of exons 7–10, primers were designed
according to the published sequence of the human
Gs protein gene (28). The primers used were as
follows: forward primer, 5'-TTC TTT TTC TCC CAG CTT CCT-3'; and reverse
primer, 5'-GGT TGG TCT GGT TGT CCT CC-3'. For sequencing of the PCR
products, M21-13 and M13-tails were added to all forward and reverse
primers, respectively.
PCR was performed in a 50-µL reaction mixture containing 100 ng genomic DNA, 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.01% gelatin, 200 µmol/L deoxy-NTP, 1 U PrimeZyme polymerase (Biometra, Gottingen, Germany), and 10 pmol of each primer. After an initial denaturation of 3 min at 95 C, samples were subjected to 30 cycles of 30 s at 95 C, 30 s at 56 C (54 C for amplification of exon 9, 52 C for amplification of exons 7–10 of the gsp gene), and 1 min at 72 C, followed by a final extension step of 6 min at 72 C. PCR products were purified by polyethylene glycol precipitation (29). Sequencing of PCR products was performed with universal dye primers (Applied Biosystems, Weiterstadt, Germany) and Thermosequenase (Amersham, Braunschweig, Germany). Both strands were sequenced. Sequencing reactions were analyzed on an automatic sequencer (ABI 373 A, PE Applied Biosystems, Foster City, CA). In the case of TTNs without TSHR mutations in exon 10, a second DNA extraction, PCR, and sequencing reaction was performed.
Allele-specific PCR
An allele-specific PCR was designed for mutations at codon 201
(ArgHis, Cys; exon 8) and codon 227 (GlnHis, Arg: exon 9). For
codon 201, the forward primers were as follows: 5'-CCT GCT TCG CTG
CCG-3' (wild-type) and 5'-CCT GCT TCG CTG CCA-3' (ArgHis), and
5'-ACC TGC TTC GCT GCC-3' (wild-type) and 5'-ACC TGC TTC GCT GCT-3'
(ArgCys). The reverse primer was 5'-GGT TGG TCT GGT TGT CCT CC-3'.
For codon 227, the forward primer was 5'-TTC TTT TTC TCC CAG CTT
CCT-3'. The reverse primers were 5'-CGG CGT TCA TCG CGC-3' (wild-type)
and 5'-CGG CGT TCA TCG CGA-3' (GlnHis), and 5'-GGC GTT CAT CGC
GCT-3' (wild-type) and 5'-GGC GTT CAT CGC GCC-3' (GlnArg).
Conditions of the allele-specific PCR were identical to the protocol
for amplification of exons 7–10 of the gsp gene. The
allele-specific PCR was performed in all TTNs in which neither TSHR nor
gsp mutations were detected by direct sequencing. Positive
controls with known Gs protein mutations were
included for both gsp sequencing and the allele-specific
PCR.
Cloning of new TSHR mutations
Exon 10 of the TSHR gene was amplified by PCR, using genomic DNA extracted from the TTNs with the Val656Phe and Del613–621 TSHR mutations as template. The primers for amplification were as follows: forward primer, 5'-ATC CTT GAG TCC TTG ATG TGT AAT-3'; and reverse primer, 5'-TTA CAA AAC CGT TTG CAT ATA CTC TT-3'. PCR products were cloned in pUC57 (MBI Fermentas, Vilnius, Lithuania). Resulting recombinant vectors were sequenced with Thermosequenase and dye-labeled terminators (Amersham) using the sequencing primer 5'-AAG TCC GAT GAG TCC AAC CCG-3'. Constructs containing the mutant allele were cleaved with CvnI and BstEII (positions 1604–2169). Finally, the mutated TSHR constructs were generated by replacing the CvnI-BstEII segment in the wild-type receptor cloned in pSVl (13) with the corresponding mutated segment cloned in pUC57.
Expression of mutated TSHR constructs
For transient expression in COS-7 cells, the constructs were transfected in 100-mm dishes with 6 µg DNA of wild-type or mutated receptor constructs using the diethylaminoethyl-dextran method (30). Twenty-four hours after transfection, the cells were split and plated in six-well plates. Forty-eight hours after transfection, the cells were used for stimulation and detection of cAMP. Three 30-mm dishes were prepared for each condition.
Measurement of cAMP
Transfected cells (4 x 105/well) were washed with serum-free DMEM without antibiotics after preincubation for 30 min with the same medium containing 1 mmol/L isobutylmethylxanthine (IBMX). Subsequently, the cells were incubated with or without bovine TSH (100 mU/mL; Sigma Chemcial Co., St. Louis, MO) for 60 min in the presence of 1 mmol/L IBMX. Thereafter, the medium was removed, and 1 mL 0.1 N HCl was added. cAMP was measured in the cell extracts with a commercial kit (Amersham) according to the manufacturer’s instructions. The results from a representative experiment are expressed as the mean cAMP values ± SE per 30-mm dish.
Binding assays
Transfected cells (4 x 105/well) were
washed once with Hanks’ solution without NaCl containing 280 mmol/L
sucrose, 0.2% BSA, and 2.5% low fat milk (4). Thereafter, the cells
were incubated in the same medium in the presence of 130,000 counts/min
[125I]TSH (TRAK Assays, BRAHMS Diagnostica, Berlin,
Germany; 25 µCi/µg; 40 U/mg) and the appropriate concentrations of
cold TSH at room temperature for 4 h. Before the cells were
solubilized with 1 N NaOH, they were washed twice with
Hanks’ solution. The bound radioactivity was determined in a
-counter. TSH or TSHR concentrations were expressed in milliunits
per mL. The data were analyzed assuming a 1:1 stoichiometry for TSH
binding to its receptor using the fitting module (31, 32) of SigmaPlot
2.0 for Windows (Jandel Scientific GmbH, Erkrath, Germany).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table 2). Moreover, one toxic nodule harbored a
TSHR mutation in the extracellular domain. This TSHR mutation affects
residue 281 in exon 9 (Ser281Asn) and has only very
recently been reported by other investigators (17). These mutations
have previously been characterized and have been shown to confer
constitutive activity upon the TSHR (1, 12, 13, 17, 33).
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). Secondly, a
27-bp deletion was identified in the third intracellular loop. This
mutation results in a deletion of nine amino acids at codons 613–621
without creating a frame shift or a stop codon (Fig. 3).
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). Compared to the
wild-type TSHR, the deletion (Del 613–621) displayed a 5- to 6-fold
increase in basal cAMP levels (14.3 ± 0.14 pmol/well) and a
markedly blunted response (17.55 ± 2.2 pmol/well) to maximal TSH
stimulation (Fig. 4).
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Moreover, a TSHR polymorphism (GACGAG) changing aspartic acid for
glutamic acid at codon 727 in the carboxy-terminus was identified in
TTNs and adjacent normal thyroid tissue of eight patients (including
four patients with a somatic TSHR mutation). This TSHR polymorphism has
previously been reported (34).
In the 16 TTNs, in which no somatic TSHR mutations in exon 9 and 10 were found, subsequent screening for mutations in exons 7–10 of the gsp gene was performed. However, no gsp mutations were identified by direct sequencing of these exons in any of the 16 TTNs. To confirm the absence of gsp mutations at the previously described mutational hot spots at codons 201 and 227 (2, 15, 17, 18), an allele-specific PCR was performed for the mutations Arg201His/Cys and Gly227His/Arg. In accordance with the results obtained by direct sequencing of exons 7–10 of the gsp gene, no PCR products were found with the mutation amplification primers in any of the 16 TTNs (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Including our study there are four different reports on TSHR frequency in TTNs determined by direct sequencing of the entire exon 10 of the TSHR. Although we found a prevalence of 45% in exon 10 (14 of 31), the frequency of TSHR mutations in exon 10 was reported to be 20% (9 of 44) (16), 67% (16 to 24) (17), and 82% (9 of 11) (12) in the other studies. Apart from the aspect that the highest mutation frequency was detected in the smallest series of samples, the reasons for this discrepancy in TSHR mutation prevalence are not immediately apparent.
Among the possible explanations to be considered is that the choice of sequencing enzymes such as Taq polymerase (Promega, M Medical Genenco, Florence, Italy) (16), Sequenase version 2.0 (U.S. Biochemical Corp., Cleveland, OH) (12), or Thermo sequenase (this study, 35) may affect the sensitivity for detection of heterozygous point mutations. Sequencing of further extracellular exons changes the percentage of hot nodules with TSHR mutations, as demonstrated by the identification of constitutively activating mutations in exon 9, leading to prevalences of 75% and 48% (Ref. 17 and our study). Therefore, the limitation of most studies to exon 10 screening might be an additional reason for discrepancies in mutation prevalences.
Although the frequency of gsp mutations in TTNs was previously reported to be 4–38% (2, 15, 16, 17, 18), we did not find mutations in exons 7–10 of the gsp gene in 31 TTNs. Two other studies have investigated the prevalence of TSHR and gsp mutations in the same series of TTNs. In one study, only 1 of 24 TTNs (4%) harbored a gsp mutation (17), which, in accordance with our results, argues for an only minor role of these mutations in the etiopathogenesis of TTNs. However, in the other study, a gsp mutation frequency of 24% was reported (15, 16). It is noteworthy that mutation screening in the latter study was performed by oligonucleotide probing. Other investigators employing similar methods also reported a high gsp mutation frequency of 25% (2) to 38% (18) in TTNs, although on a considerably smaller series of samples. By comparison with oligonucleotide probing, detection of heterozygous somatic mutations by direct sequencing requires a distribution of mutated and wild-type alleles close to 1:1. Tissue of TTNs can be contaminated with blood or stroma cells, which will reduce the relative frequency of mutated alleles, thus eventually making their detection by direct sequencing difficult or even impossible. In contrast, allele-specific PCR is more sensitive in detecting mutated alleles independently of the wild-type allele ratio. Therefore, the absence of PCR products with the allele-specific amplification primers for mutations at gsp codons 201 and 227 strongly argues for the absence of gsp mutations in these positions in our series.
Identification of new TSHR mutations
The identification of a new constitutively activating mutation at codon 656 provides further evidence for involvement of the third extracellular TSHR loop in TSH-independent adenylyl cyclase activation. As the Val656Phe mutation displays a 4- to 5-fold increase in basal cAMP levels, it is a mutation with a relatively high constitutive activity. In contrast, the other hitherto identified point mutation in the third extracellular TSHR loop (Asn650Tyr) shows only a 2-fold increase in basal cAMP levels (4).
In accordance with results from other studies (12, 16, 17), the distribution pattern of TSHR mutations in our series favors the third intracellular loop and the sixth transmembrane segment as mutational hot spots, suggesting that these regions are particularly important for maintaining the receptor in a silent state. In this context it appears of great interest that the deletion of nine amino acids (Del 613–621) in the third intracellular TSHR loop also leads to constitutive activation of the receptor. Two other deletions have previously been identified in exon 10 of the TSHR. In the case of the single amino acid deletion at codon 619 combined with an amino acid substitution at codon 620 (Thr620Ser), no constitutive activity was reported (19), whereas the recently identified deletion of four amino acids in the third extracellular loop (Del 658–661) displayed constitutive activity (17). Most interestingly, the new deletion mutation (Del 613–621) identified in our study showed increased basal cAMP levels to the same extent as previously characterized TSHR point mutations in the third intracellular TSHR loop, e.g. the Asp619Gly and the Ala623Val mutations. Yet, although in the case of the latter mutations an at least 2-fold increase in cAMP levels in response to TSH stimulation has been reported (1, 13), cAMP levels for the deletion mutation (Del 613–621) were only marginally raised in response to maximal TSH stimulation.
Etiology of mutation-negative TTNs
The pathogenesis of mutation-negative hyperfunctioning thyroid nodules is a challenging issue. As the dominant clinical aspects of TTNs, e.g. hyperthyroidism and nodule growth, could be explained by activation of the adenylyl cyclase cascade (36, 37), one is easily inclined to look for other mutations in this signal transduction pathway. Firstly, mutations may be localized in domains of the TSHR or gsp genes that have not been investigated, i.e. more proximal parts of the amino-terminal extracellular TSHR domain and other than exons 7–10 of the gsp gene. Even though the major function of the extracellular TSHR domain is currently defined by its role in TSH binding, the identification of up to five different constitutively activating mutations in the extracellular loops of the transmembrane domain (Refs. 4, 12, and 17 and our study) and the finding to date of two constitutively activating TSHR mutations in exon 9 (Ref. 17 and our study) indicate that the ability for TSH-independent activation of the cAMP cascade may not be exclusively restricted to the transmembrane and intracellular TSHR parts. Secondly, overstimulation of cAMP production could also result from simple overexpression of the wild-type receptor, the rationale being that even the nonmutated TSHR displays considerable basal activity (38). Thirdly, activating mutations further downstream in the cAMP cascade, e.g. in the adenylyl cyclase gene, may be involved. Fourthly, it may be hypothesized that mutations with inactivating properties, e.g. in the Gi proteins or in TSHR promoter-repressing genes, could result in the same biological effects as the hitherto described constitutively activating mutations.
Heterozygous point mutations in the ras gene have been identified in TTNs (15, 39). As increased basal cAMP levels have not been demonstrated in all TTNs, possibly because of methodological obstacles (40), alterations in other signal transduction pathways may also have to be considered. Moreover, mutations in and/or increased expression of protein kinase C (41) have been reported in thyroid tumors, yet it has not been elucidated how and whether these alterations could contribute to the clinical hallmarks of TTNs.
For further insight into the pathogenesis of thyroid autonomy, clarification of the molecular pathogenesis of TSHR and gsp mutation-negative TTNs, which have been reported with varying frequency in all studies, is definitely required.
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Acknowledgments |
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Footnotes |
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Received December 18, 1996.
Revised July 3, 1997.
Accepted July 25, 1997.
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X. De Deken, C. Vilain, J. Van Sande, J. E. Dumont, and F. Miot Decrease of Telomere Length in Thyroid Adenomas without Telomerase Activity J. Clin. Endocrinol. Metab., December 1, 1998; 83(12): 4368 - 4372. [Abstract] [Full Text]
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A. N. Abell, D. J. McCormick, and D. L. Segaloff Certain Activating Mutations within Helix 6 of the Human Luteinizing Hormone Receptor May Be Explained by Alterations That Allow Transmembrane Regions to Activate Gs Mol. Endocrinol., December 1, 1998; 12(12): 1857 - 1869. [Abstract] [Full Text]
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H. Biebermann, T. Schöneberg, A. Schulz, G. Krause, A. Grüters, G. Schultz, and T. Gudermann A conserved tyrosine residue (Y601) in transmembrane domain 5 of the human thyrotropin receptor serves as a molecular switch to determine G-protein coupling FASEB J, November 1, 1998; 12(14): 1461 - 1471. [Abstract] [Full Text]
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P. Wonerow, T. Schoneberg, G. Schultz, T. Gudermann, and R. Paschke Deletions in the Third Intracellular Loop of the Thyrotropin Receptor. A NEW MECHANISM FOR CONSTITUTIVE ACTIVATION J. Biol. Chem., April 3, 1998; 273(14): 7900 - 7905. [Abstract] [Full Text] [PDF]
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H.-P. Holzapfel, D. Fuhrer, P. Wonerow, G. Weinland, W. A. Scherbaum, and R. Paschke Identification of Constitutively Activating Somatic Thyrotropin Receptor Mutations in a Subset of Toxic Multinodular Goiters J. Clin. Endocrinol. Metab., December 1, 1997; 82(12): 4229 - 4233. [Abstract] [Full Text] [PDF]
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C. Govaerts, A. Lefort, S. Costagliola, S. J. Wodak, J. A. Ballesteros, J. Van Sande, L. Pardo, and G. Vassart A Conserved Asn in Transmembrane Helix 7 Is an On/Off Switch in the Activation of the Thyrotropin Receptor J. Biol. Chem., June 15, 2001; 276(25): 22991 - 22999. [Abstract] [Full Text] [PDF]
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