klinische charakterisierung von tsh-rezeptormutationen · tsh thyreoidea stimulierendes hormon . 3...

Klinische Charakterisierung von TSH-Rezeptormutationen

Dissertation

zur Erlangung des akademischen Grades

Dr. med.

an der Medizinischen Fakultät

der Universität Leipzig

eingereicht von

Julia Cordula Lüblinghoff

geb.:08.07.1983 in Iserlohn, Deutschland

angefertigt am

Universitätsklinikum Leipzig

Medizinische Klinik und Poliklinik für Endokrinologie und Nephrologie

Liebigstraße 20, D – 04103 Leipzig

betreut von

Prof. Dr. med. R. Paschke

Beschluss über die Verleihung des Doktorgrades vom 18.09.2012

Inhaltsverzeichnis

I. Bibliographische Zusammenfassung 1

II. Abkürzungsverzeichnis 2

III. Einführung 3-10

IV. Publikationen 11-26

V. Zusammenfassung 27-31

VI. Literaturverzeichnis 32-39

VII. Anlagen

Tabelle 1.1 und 1.2

Abbildung 2

Tabelle A

40-45

41-42

43-44

45

VIII. Selbstständigkeitserklärung 46

IX. Lebenslauf 47

X. Publikationsliste 48

XI. Danksagung 49

1

I. Bibliographische Beschreibung

Lüblinghoff, Julia Cordula


Universität Leipzig, Dissertation

49 S., 96 Lit., 4 Abb., 3 Tab., 4 Anlagen

Referat

Diese Dissertation untersucht einen möglichen Zusammenhang zwischen dem beschrie-

benen klinischen Verlauf bei Patienten mit konstitutiv aktivierenden TSH-

Rezeptormutationen und der gemessenen in vitro Aktivität.

Konstitutiv aktivierende Mutationen finden sich als somatische Mutationen in autonomen

Adenomen und als Keimbahnmutationen bei Patienten mit sporadischer bzw. familiärer

nicht-autoimmuner Hyperthyreose. Die in vitro Aktivität der zu Grunde liegenden TSH-

Rezeptormutationen wird mit Hilfe der Linearen Regressions-Analyse bestimmt. Dies ist

ein Verfahren, welches die basale Produktion des second messenger cAMP (Cyclo-

Adenosinmonophosphat) misst, unter Berücksichtigung der Expression des TSH-

Rezeptors.

Die Analyse der Krankheitsverläufe der sporadischen nicht-autoimmunen Hyperthyreose

zeigt keinen eindeutigen Bezug zur gemessenen in vitro Aktivität. Es besteht jedoch eine

höhere in vitro Aktivität bei Mutationen, die sowohl bei der nicht-autoimmunen sporadi-

schen Hyperthyreose und in autonomen Adenomen zu finden sind, im Vergleich zu aus-

schließlich familiären Mutationen. Dies entspricht auch dem klinischen Eindruck. Für die

wenigen bekannten Fälle der sporadischen nicht-autoimmunen Hyperthyreose wurden

dramatische Verläufe mit häufigen Rückfällen unter medikamentöser Therapie und zahl-

reichen Komplikationen (z.B. mentale Retardierung, Kraniosynostose, zerebrale Ventriku-

lomegalie, beschleunigte Knochenreifung) beschrieben.

2

II. Abkürzungsverzeichnis

AA Autonome Adenome

cAMP Cyclo-Adenosinmonophosphat

cDNA komplementäre DNA

COS-7 Zelllinie Fibroblasten-Zelllinie, die für Transfektionsexperimente

verwendet wird

Del Deletion

GDP Guanosindiphosphat

GTP Guanosintriphosphat

FACS Fluorescence Activated Cell Sorting

FNHA familiäre nicht-autoimmune Hyperthyreose 125I bTSH mit radioaktivem Iod markiertes bovines TSH

IP Inositolphosphat

NAH nicht-autoimmune Hyperthyreose

PCR Polymerase-Kettenreaktion

SNAH sporadische nicht-autoimmune Hyperthyreose

TSH Thyreoidea stimulierendes Hormon

3


III. Einführung

Aufbau und Funktion des TSH-Rezeptors

Die Regulation der Schilddrüsenfunktion und des Schilddrüsenwachstums erfolgt über die

Bindung des TSH (Thyreoida stimulierendes Hormon) an die extrazelluläre Domäne des

TSH-Rezeptors. Dieser gehört zur Familie der G-Protein gekoppelten Rezeptoren und

setzt sich aus einer N-terminalen extrazellulären Domäne, sieben Transmembrandomä-

nen, welche durch drei extra- und drei intrazelluläre Schleifen miteinander verbunden

sind, und aus einem intrazellulären C-Terminus zusammen (Abbildung 1, S. 4). Die Bin-

dung des TSH an die extrazelluläre Domäne aktiviert den Rezeptor. An der �-Untereinheit

des G-Proteins kommt es zum Austausch von GDP (Guanosindiphosphat) gegen GTP

(Guanosintriphosphat) und damit zur Dissoziation des G-Proteins in die �-Untereinheit

und in die �- und �-Untereinheit. Über die �s-Untereinheit wird die cAMP-Kaskade (Cyclo-

Adenosinmonophosphat) stimuliert (81). Es erfolgt zum einen die funktionelle Aktivierung

der Schilddrüse, welche in einer hyperthyreoten Stoffwechsellage Ausdruck findet und

zum anderen die Proliferationsaktivierung der Thyreozyten, welche sich in einer

Schilddrüsenhyperplasie äußert. Bei vielfach höheren TSH-Konzentrationen (Faktor 5-10)

ist der TSH-Rezeptor in der Lage neben G�s auch G�q zu aktivieren. Dies bewirkt über

die Stimulation der Inositolphosphat-Kaskade die Proliferation der Thyreozyten (22; 81;

93).

4

Hyperthyreose

�s �q

�

�

��

GTP GTP

TSH-Rezeptor

Zellmembran

TSHN

Proliferation der Thyreozyten

Proliferation der Thyreozyten

Aktivierung der Phospholipase, IP-Anstieg (bei hoher TSH-Konzentration)

Aktivierung derAdenylatcyclase, cAMP-Anstieg

C �

Abbildung 1: TSH-Rezeptor und G-Protein gekoppelte Aktivierung (vereinfachte Darstellung)

Der TSH-Rezeptor ist in der Zellmembran verankert und an das G-Protein gekoppelt. Er setzt sich

aus einer N-terminalen extrazellulären Domäne, sieben Transmembrandomänen, welche durch

drei extra- und drei intrazelluläre Schleifen miteinander verbunden sind, und aus einem intrazellulä-

ren C-Terminus zusammen. Über das heterotrimere G-Protein, bestehend aus der �-, �- und �-

Untereinheit, wird die Signaltransduktion vermittelt. Die Bindung des TSH (Thyreoidea stimulieren-

des Hormon) an die extrazelluläre Domäne aktiviert den Rezeptor. An der �-Untereinheit kommt es

zum Austausch von GDP (Guanosindiphosphat) gegen GTP (Guanosintriphosphat). Die

�-Untereinheit dissoziiert von der �- und �-Untereinheit. Die �s-Untereinheit aktiviert die Adenylat-

cyclase und es kommt zum Anstieg des cAMP (Cyclo-Adenonsinmonophophat). Das cAMP bewirkt

als intrazellulärer Botenstoff zum einem die funktionelle Aktivierung der Schilddrüse, welches zur

hyperthyreoten Stoffwechsellage führt und zum anderen die Proliferationsaktivierung der Thyreozy-

ten, welches zu einer Schilddrüsenhyperplasie führt. Bei vielfach höheren TSH-Konzentrationen

(Faktor 5-10) ist der TSH-Rezeptor in der Lage neben G�s auch G�q zu aktivieren. Dies führt über

die Aktivierung der Phospholipase zum IP-Anstieg (Inositolphosphat) und damit zur Stimulation der

Thyreozytenproliferation.

5

Konstitutiv aktivierende Mutationen

Eine TSH-unabhängige und damit permanente Aktivierung der G�s-Untereinhet des

G-Proteins findet sich bei den sog. konstitutiv aktivierenden TSH-Rezeptormutationen.

Diese Mutationen sind in autonomen Schilddrüsenadenomen (AA) und im Schilddrüsen-

gewebe von Patienten mit familiären bzw. sporadischen Formen bei der nicht-

autoimmunen Hyperthyreose (NAH) identifiziert worden. Bei den autonomen Adenomen

lassen sich somatische Mutationen im Knotengewebe, jedoch nicht im umgebenden ge-

sunden Schilddrüsengewebe nachweisen. Bei den Patienten mit der familiären bzw. spo-

radischen Form der nicht-autoimmunen Hyperthyreose wurden Keimbahnmutationen

identifiziert, die dazu führen, dass jede Schilddrüsenzelle die Mutation exprimiert. Die

Keimbahnmutationen treten bei der sporadischen nicht-autoimmunen Hyperthyreose

(SNAH) als de novo Mutationen auf bzw. als autosomal dominante Mutationen bei der

familiären nicht-autoimmunen Hyperthyreose (FNAH) (69).

Erstbeschreibung der Mutationen

Im Jahr 1993 wurde von Parma et al. (66) erstmals eine konstitutiv aktivierende somati-

sche TSH-Rezeptormutation in zwei solitären autonomen Adenomen identifiziert. Bis heu-

te konnten 45 verschiedene Punktmutationen und drei Deletionen im TSH-Rezeptor in

insgesamt 3341 verschiedenen autonomen Adenomen nachgewiesen werden (3; 15-17;

23; 27; 28; 32; 44; 46; 47; 58; 63; 65; 67; 68; 70; 72; 75; 76; 80; 82; 84; 86; 88; 89; 92; 96)

(Anlagen: Tabelle 1.1 und 1.2, Abbildung 2 S. 41-44; http://innere.uniklinikum-

leipzig.de/tsh/).

Die erste Familie mit einer nicht-autoimmunen Hyperthyreose wurde bereits 1982 von

Thomas et al. beschrieben (Nancy-Familie) (83). Diese Arbeitsgruppe beobachtete ein

gehäuftes Auftreten von Hyperthyreosen über vier Generationen bei Abwesenheit der

Stigmata einer autoimmunen Thyreopathie (Nachweis von TSH-Rezeptorantikörpern und

endokriner Orbitopathie). Die zu Grunde liegende Punktmutation im TSH-Rezeptor wurde

jedoch erst 1994 von Duprez et al. (18) detektiert; ein autosomal dominanter Erbgang die-

ser familiären Keimbahn TSH-Rezeptormutation war erkennbar. In den letzten 30 Jahren

sind 26 Familien mit 192 verschiedenen TSH-Rezeptormutationen beschrieben worden (1;

2; 4; 7; 13; 18; 19; 21; 25; 26; 34; 35; 41; 42; 51; 52; 59; 62; 64; 71; 73; 78; 79; 83; 87; 91;

95) (Anlagen: Tabelle 1.1, S. 41 ).

1 Eine Deletion und mehrere somatische TSH-Rezeptormutationen sind erst nach Veröffentlichung des zweiten Artikels dieser kumulativen Dissertation berichtet worden (58; 60). 2 Zwei Familien mit TSH-Rezeptormutationen wurden erst nach Veröffentlichung des zweiten Arti-kels dieser kumulativen Dissertation berichtet (59; 95).

6

Der erste Fall eines Patienten mit einer sporadischen nicht-autoimmune Hyperthyreose

und zu Grunde liegender de novo TSH-Rezeptormutation wurde 1996 von de Roux et al.

beschrieben (14). Bis heute sind 16 Patienten3 mit dem Krankheitsbild der sporadischen

nicht-autoimmunen Hyperthyreose bekannt (6; 8-10; 12; 14; 20; 24; 30; 33; 43; 45; 50; 61;

85; 94) (Anlagen: Tabelle A, S. 45, http://innere.uniklinikum-leipzig.de/tsh/).

Nachweis der Mutationen

Die konstitutiv aktivierenden TSH-Rezeptormutationen sind bis auf drei Ausnahmen

(Del E403, Del Y613-K621 und Del E339-G367, (23; 46; 60; 89; 96); vgl. Tabelle 1.2,

S. 42) Punktmutationen mit dem Austausch von ein oder zwei DNA-Basen. Konstitutiv ak-

tivierende TSH-Rezeptormutationen wurden bislang ausschließlich in den Exons 9 und 10

identifiziert (31). Exon 9 kodiert für einen Teil der extrazellulären Domäne, während Exon

10 die genetische Information für die Transmembrandomäne und den intrazellulären C-

Terminus des Rezeptors enthält. Der Nachweis der somatischen und der Keimbahn TSH-

Rezeptormutationen erfolgt deshalb durch direkte Sequenzierung der Exons 9 und 10.

Hierfür wird DNA aus dem Schilddrüsengewebe extrahiert, mit Hilfe der PCR (Polymera-

se-Kettenreaktion) amplifiziert und anschließend sequenziert. Zur Differenzierung zwi-

schen somatischer und Keimbahnmutation wird zusätzlich die aus peripheren Blutleuko-

zyten extrahierte DNA analysiert. Beim Vorliegen einer Keimbahnmutation lässt sich auch

hier die jeweilige TSH-Rezeptormutation nachweisen (90).

3 Zwei Patienten (I630L, Bertalan et al., 2010 (6) und I486N, Biebermann et al., 2011 (8)) wurden erst nach Veröffentlichung des ersten Artikels dieser kumulativen Dissertation beschrieben.

7

Funktionelle Charakterisierung der TSH-Rezeptormutationen, Definition der

konstitutiven Aktivität

Für die funktionelle Charakterisierung der TSH-Rezeptormutationen wird die cDNA (kom-

plementäre DNA) des Wildtyp TSH-Rezeptors als Template verwendet, welcher in einem

eukaryotischen Expressionsvektor kloniert vorliegt. Basierend auf diesem Template wird

mittels ortsgerichteter Mutagenese der gewünschte Aminosäureaustausch auf Ebene der

cDNA eingeführt. Der mutierte TSH-Rezeptor wird dann mit Hilfe des Expressionsvektors

in einer geeigneten Zelllinie (meist COS-7 Zellen) exprimiert. Es erfolgt die Messung des

second messenger cAMP (Cyclo-Adenosinmonophosphat), der bei der rezeptorspezifi-

schen Aktivierung der G�s-Signalkaskade intrazellulär akkumuliert. Typisch für die konsti-

tutiv aktivierenden Mutationen ist, dass auch ohne TSH-Stimulation eine basale (konstitu-

tive) Aktivität messbar ist (55). Da auch der Wildtyp TSH-Rezeptor eine leichte konstituti-

ve Aktivität aufweist (11), wird die basale cAMP-Akkumulation des Wildtyp TSH-

Rezeptors gleich 1 gesetzt. Außerdem besteht zwischen der basalen Rezeptoraktivität

und der Rezeptorexpression an der Zelloberfläche ein lineares Verhältnis. Aus diesem

Grund muss beim Vergleich der gemessenen basalen Rezeptoraktivität mit dem Wildtyp

TSH-Rezeptor auch die Zahl der an der Zelloberfläche exprimierten Rezeptoren mit ein-

bezogen werden. Dieser Sachverhalt wird bei der Bestimmung der LRA (Lineare Regres-

sions-Analyse) berücksichtigt. Bei dieser Methode der in vitro Aktivitätsmessung werden

parallel die basale cAMP-Produktion und die TSH-Rezeptorexpression gemessen, indem

die untersuchte Zellreihe mit ansteigenden DNA-Konzentrationen transfiziert wird. Die

TSH-Rezeptorexpression wird mittels FACS Analyse (Fluorescence Activated Cell Sor-

ting) oder mittels Bindung des radioaktiv markierten 125I-bTSH (bovine TSH) quantifiziert

(56). Zur Bestimmung der LRA werden dann die Werte der basalen cAMP-Akkumulation

des Mutationsvektors abzüglich der basalen cAMP-Akkumulation des leeren Expressions-

vektors (y-Achse) als Funktion der Mutationsvektorexpression abzüglich der Expression

des leeren Expressionsvektors aufgetragen (x-Achse). Es zeigt sich ein linearer Anstieg

der basalen cAMP-Akkumulation für jede untersuchte TSH-Rezeptormutation. Die Stei-

gung dieser Geraden (= slope-Wert) ergibt den LRA-Wert. Um die LRA-Werte der einzel-

nen Mutationen vergleichen zu können, wird der slope-Wert des Wildtyp TSH-Rezeptors

mit 1 gleich gesetzt (56). Abbildung 3 (nächste Seite) zeigt die gemessenen Werte und

die errechneten slope-Werte für die TSH-Rezeptormutationen V597F und I568V (55).

8

0 50 100 150 200 250 300 350 4000

10

20

30

40

wt = 1

I568V = 4.1±±±±1.1

V597F = 23.8±±±±2.6

slope

Arbitrary fluorescence units

cA

MP

[n

M]

Abbildung 3: Bestimmung der konstitutiven Aktivität mit Hilfe der Linearen Regressions-Analyse

Beispiel für die Bestimmung von drei slope-Werten (= Steigung der Regressionsgeraden) für die

TSH-Rezeptormutationen V597F und I568V im Vergleich zum Wildtyp TSH-Rezeptor (wt).

COS-7 Zellen wurden mit ansteigenden DNA-Konzentrationen transfiziert. Die konstitutive Aktivität

ergibt sich aus der basalen cAMP-Akkumulation (y-Achse) als Funktion der Rezeptorexpression

(x-Achse), welche mittels FACS Analyse bestimmt wurde. Die slopes wurden mit Hilfe der Linearen

Regressions-Funktion von Graph Pad 4.0 für Windows errechnet. Um die Mutationen untereinan-

der vergleichen zu können, wird der slope des Wildtyp TSH-Rezeptors mit 1 gleich gesetzt (55).

Die Relevanz der Methode der LRA-Bestimmung zeigt sich dadurch, dass ohne Berück-

sichtigung der Rezeptorexpression TSH-Rezeptormutationen fälschlicherweise als konsti-

tutiv aktivierend eingestuft wurden, da bei den bisherigen Untersuchungen oft nur die ba-

sale cAMP-Akkumulation unabhängig von der TSH-Rezeptorexpression untersucht wur-

de. In der Arbeit von Jäschke et al. (39) wurden fünf TSH-Rezeptormutationen, denen

man bislang eine konstitutive Aktivität zugeschrieben hatte, erneut untersucht. Es zeigte

sich, dass nur eine der untersuchten Mutationen (F631I) tatsächlich konstitutiv aktiv war.

Die familiäre TSH-Rezeptormutation I691F (52) und die somatische TSH-

Rezeptormutation F666L (82) hingegen zeigten keine konstitutive Aktivität.4

Die LRA-Bestimmung ist bislang die einzige direkte experimentelle Methode, mit der sich

die TSH-Rezeptormutationen unabhängig von der Rezeptorexpression vergleichen lassen

(56) und bildet somit die Grundlage für Vergleiche der in vitro Aktivitäten der einzelnen

Mutationen untereinander.

4 Zur Vollständigkeit sind diese Mutationen in Tabelle 1.1 und 1.2. aufgeführt.

9

Ableitung der Rationale

Da die Bestimmung der LRA eine neue Methode zur Charakterisierung der in vitro Aktivi-

tät der konstitutiv aktivierenden Mutationen ist und für einen Großteil der Mutationen die

LRA-Werte erst kürzlich publiziert (40; 54; 55; 57; 76), bzw. im Rahmen dieser Arbeit be-

stimmt wurden (LRA-Werte für M453T, L512Q, I568T, F631L, T632I, D633Y; Tabelle A,

S. 45), gibt es bislang keine umfassende Analyse der LRA-Werte in Zusammenhang mit

dem Phänotyp der somatischen, sporadischen und familiären TSH-Rezeptormutationen

und keinen Vergleich der LRA-Werte zwischen den einzelnen Mutationstypen (somati-

sche, sporadische und familiäre Mutationen).

Für die 165 beschriebenen Fälle der sporadischen nicht-autoimmunen Hyperthyreose wur-

den unterschiedlichste klinische Verläufe berichtet. Kopp et al. (12) beschrieben 1995 ei-

nen Patienten mit Manifestation der Hyperthyreose im ersten Lebensmonat. Im weiteren

Verlauf fielen bei diesem Patienten eine beschleunigte Knochenreifung und eine mentale

Retardierung auf. Der Patient zeigte trotz wiederholter Thyreoidektomie einen Rückfall der

Erkrankung und war erst nach ablativer Radiojodtherapie euthyreot. Ein exzessives

Schilddrüsenwachstum (Schilddrüsen-Volumen von 370 ml im Alter von 20 Jahren) wurde

von Nishihara et al. (8) beschrieben. Die Patientin war bereits bei der Geburt hyperthyre-

ot, zeigte eine beschleunigte Knochenreifung und eine Kraniosynostose. Eine euthyreote

Stoffwechsellage wurde ebenfalls erst nach ablativer Radiojodtherapie erreicht.

Im Gegensatz zu diesen beiden dramatischen klinischen Verläufen berichteten Börgel et

al. (3) über eine Patientin mit neonataler Manifestation der Erkrankung, welche medika-

mentös gut kontrolliert werden konnte. Außer einer dezenten Schilddrüsenvergrößerung

wurden keine Krankheitsfolgen berichtet. Diese drei Krankheitsverläufe zeigen die Varia-

bilität des Krankheitsbildes der sporadischen nicht-autoimmunen Hyperthyreose (SNAH)

und werfen die Frage auf, ob der klinische Verlauf maßgeblich durch die in vitro Aktivität

der zu Grunde liegenden TSH-Rezeptormutation beeinflusst wird. Diese Fragestellung

wurde im ersten Teil dieser Arbeit untersucht (S. 12-17).

Die beschriebenen Krankheitsverläufe der Patienten mit familiären TSH-

Rezeptormutationen zeigen im Vergleich zu den sporadischen Mutationen eine Tendenz

zu milderen Krankheitsverläufen. Im Gegensatz zur SNAH, die sich meist im ersten Le-

bensmonat (elf von 16 Patienten), spätestens jedoch bis zum elften Lebensmonat mani-

festiert (24), variiert der Krankheitsbeginn bei den familiären Mutationen zwischen zwölf

Tagen (1) und 64 Jahren (59). Auch innerhalb einer Familie ist das Alter der Erstmanifes-

5 Zwei Patienten (I630L, Bertalan et al., 2010 (6) und I486N, Biebermann et al., 2011 (8)) wurden erst nach Veröffentlichung des ersten Artikels dieser kumulativen Dissertation beschrieben.

10

tation variabel. Thomas et al. (83) beschrieben eine Altersspanne von zehn bis 36 Jahren,

Duprez et al. (18) von 18 Monaten bis 53 Jahren, Führer et al. (25) von zwei bis 21 Jahren

und Karges et al. (41) von vier bis zu 50 Jahren. Insgesamt ist die Erstmanifestation aber

später als bei den sporadischen Mutationen. Nur fünf der 95 Patienten mit der familiären

nicht-autoimmunen Hyperthyreose (FNAH) zeigten Symptome im ersten Lebensmonat (1;

26; 73; 78; 79). Die klinischen Verläufe der FNAH variieren von medikamentös gut ein-

stellbaren Verläufen (51), über subklinische Hyperthyreosen (4; 62; 71; 83; 91) bis hin zu

schweren Verläufen mit beschleunigter Knochenreifung, Hepatosplenomegalie, Ikterus,

zerebraler Ventrikulomegalie, mentaler Retardierung und der Notwendigkeit wiederholter

Radiojodtherapien (79).

Die überwiegend frühe Erstmanifestation und die schweren klinischen Verläufe, der SNAH

haben wiederholt die Frage aufgeworfen, ob sporadische Mutationen eine höhere in vitro

Aktivität aufweisen als familiäre. Auf der Grundlage einer komparativen Gen-Analyse wur-

de außerdem ein stärkerer Effekt der somatischen TSH-Rezeptormutationen in autono-

men Adenomen verglichen mit den familiären TSH-Rezeptormutationen (31) vermutet. Bis

heute gibt es keine Korrelationsanalyse der Phänotypen der autonomen Adenome (AA)

mit der in vitro Aktivität und außer bei der jodinduzierten Hyperthyreose (77) sind die Ur-

sachen für die große Variabilität der deutlich unterschiedlichen klinischen Verläufe der Pa-

tienten mit AA nicht weiter definiert worden. Im zweiten Teil dieser Arbeit (S. 18-26) wurde

deshalb einerseits nach signifikanten Unterschieden der LRA-Werte der sporadischen,

familiären und somatischen Mutationen gesucht. Andererseits wurde eine Korrelation kli-

nischer Merkmale der Patienten mit autonomen Adenomen mit den entsprechenden LRA-

Werten durchgeführt.

11

IV. Publikationen

1. LÜBLINGHOFF J, MÜLLER S, SONTHEIMER J AND PASCHKE R

„Lack of Consistent Association of TSH Receptor Mutations In Vitro Activity with the Clini-

cal Course of Patients with Sporadic Non-autoimmune Hyperthyroidism.“

Journal of Endocrinological Investigation. 2010 33: 228-33.

2. LUEBLINGHOFF J, ESZLINGER M, JAESCHKE H, MUELLER S, BIRCAN R, GOZU H, SANCAK S,

AKALIN S AND PASCHKE R

„Shared sporadic and somatic thyrotropin receptor mutations display more active in vitro

activities than familial thyrotropin receptor mutations.”

Thyroid. 2011 21:221-9

228

J. Endocrinol. Invest. 33: 228-233, 2010DOI: 10.3275/6476

ABSTRACT. Background: Up to date, 14 patients with spo-radic non-autoimmune hyperthyroidism (SNAH) caused bysporadic germline mutations in the TSH receptor (TSHR)gene have been reported. Despite considerable differencesin the activity of hyperthyroidism, all SNAH case reports con-cluded that the demonstrated constitutive activity explainsthe phenotype. Aim: Recently, linear regression analysis(LRA) of constitutive activity as a function of TSHR expres-sion determined by 125I-bTSH binding or fluorescence acti-vated cell sorting analysis was described as a more reliableway of characterizing the in vitro activity (IVA) of a consti-tutively activating TSHR mutation. Therefore, we analyzed apossible genotype-phenotype correlation in a systematic re-view of the case reports and investigated the TSHR muta-tion’s LRA in selected cases. Material and methods: We de-termined the LRA for all sporadic germline mutations whichhad not previously been reported. Moreover, we systemat-ically evaluated all case reports of SNAH for evidence of an

association of the clinical course (CC) with the IVA of themutated TSHR. Results: The LRA determined were: M453T(5.2±0.8), L512Q (4.5±0.7), I568T (25.6±6.3), F631L(45.9±9.4), T632I (14.5±2.7), D633Y (16.4±6.4). None of the10 examined clinical signs showed a significant associationwith the LRA. Moreover, the comparison of the CC of pa-tients harboring the same mutation (S281N, M453T, I568T,S505N) also showed no relation of the clinical activity witha high LRA. Conclusion: Considering the different diagnos-tic circumstances, therapeutic strategies and the limitationsof a systematic analysis of case reports due to the restrict-ed number of case reports and limited follow-up we foundno consistent relation of the TSHR mutation’s IVA deter-mined by LRA with the CC of patients with SNAH. This mayalso be due to the action of genetic, epigenetic, and envi-ronmental modifiers.(J. Endocrinol. Invest. 33: 228-233, 2010)©2010, Editrice Kurtis

INTRODUCTION

Constitutively activating TSH receptor (TSHR) germlinemutations have been identified as a molecular cause ofcongenital hyperthyroidism (HT). Up to date, 14 case re-ports of sporadic non-autoimmune HT (SNAH) caused bysporadic germline mutations in the TSHR gene have beenpublished (http://innere.uniklinikum-leipzig.de/tsh/)(Table 1) (1-14). The clinical courses (CC) of theses pa-tients showed considerable differences for the activity ofHT and a pronounced intrafamilial phenotypic variabilitywas also noticed early in the description of patients withfamilial non-autoimmune HT. However, all SNAH case re-ports concluded that the demonstrated constitutive ac-tivity explains the phenotype.Recently, linear regression analysis (LRA) of constitutiveactivity as a function of TSHR expression determined by125I-bTSH binding or fluorescence-activated cell sorting(FACS) analysis compared to the wild type (wt) TSHR wasdescribed as a more reliable way of characterizing the invitro activity (IVA) of a constitutively activating TSHR mu-tation (17, 30-32). Therefore, we determined the LRA for

all sporadic germline mutations which had not previous-ly been reported. Moreover, we systematically reviewedall case reports of SNAH for evidence of a possible as-sociation of the CC with the IVA of the mutated TSHR.Based on these data, we then investigated the questionif there is an association of the different sporadic TSH re-ceptor germline mutations LRA values with the respec-tive different clinical HT activity signs and findings.

MATERIAL AND METHODS

For the investigation of a possible association of the clinical andthe IVA we systematically screened the case reports for age atonset of HT, onset, morphology, and growth of goiter, prema-turity, mental retardation, craniosynostosis, low birth weight, ad-vanced bone age, and the CC characterized by the duration ofHT and the treatment by thyroid surgery or radio-iodine therapy.The metric variables “age at onset of HT”, “age at onset of goi-ter”, and “duration of HT” were analyzed for their correlationswith the LRA by a bivariate non-parametric correlation analysiscarried out with SPSS 15.0 for Windows (Spearman correlationcoefficient).All further evaluated clinical characteristics were categorical ornominal variables. For the latter, we therefore performed a testfor association (Fisher’s Exact Test) of the clinical parameterswith the LRA values of the different mutations which were clas-sified as high or low IVA according to their LRA results. As themedian of the LRA values was 15.1 (min=4.5, max=125.2), LRAvalues <15.1 were classified as low and LRA values ≥15.1 wereclassified as high IVA (Table 1).Furthermore, we tested for differences in the LRA activity be-

Key-words: Gain-of-function mutation, linear regression analysis, non-autoimmunehyperthyroidism, sporadic germline mutation, TSH-receptor mutation.

Correspondence: R. Paschke, MD, University of Leipzig III Medical Department,Philipp-Rosenthal-Str. 27, D- 04103 Leipzig, Germany.

E-mail: [email protected]

Accepted June 17, 2009.

First published online July 28, 2009.

Lack of consistent association of thyrotropin receptor mutationsin vitro activity with the clinical course of patients with sporadicnon-autoimmune hyperthyroidismJ. Lueblinghoff, S. Mueller, J. Sontheimer, and R. Paschke

III Medical Department, University of Leipzig, Leipzig, Germany

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actionaction ofof genetic,genetic, epigenetic,epigenetic, andmodifiers.

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Correlation of sporadic TSHR mutations

229

tween groups of clinical signs using non-parametric tests [Mann-Whitney-U-Test for 2 categories (e.g. prematurity: prematurebirth or birth at term) and Kruskal-Walis-Test for 3 categories(onset of HT: fetal, neonatal or late onset)].Generation of mutants, cell culture, and transfection were per-formed as previously published (22, 23). LRA of basal activity asa function of TSHR expression determined by FACS analysis wasperformed as previously published (22, 23). The results for cellsurface expression (FACS) and basal cAMP accumulation at var-ious DNA concentrations were used to calculate the constitu-tive activity of the mutants. The initial protocol to evaluate basalactivity independently from the cell surface expression was pub-lished by Hjorth et al. (33). This method was modified by Balles-teros et al. (34) for the the b2-adrenergic receptor, by Srinivasanet al. (35), for the melanocortin-4 receptor and for the TSHR bye.g. Vlaeminck-Guillem (30); Jaeschke et al. (31); Gozu et al. (17);Kleinau et al. (32).The effect of expression level on basal cAMP accumulation wasdetermined by plotting cell surface expression measured byFACS (x-axis) vs basal cAMP accumulation (y-axis) using the lin-ear regression function of GraphPad Prism 4.03 for Windows.The slope of the wt TSHR was set at 1 and slopes of the mu-tants were calculated according to this. The LRA were deter-mined for M453T, L512Q, I568T, F631L, and T632I.

RESULTS

Functional characterization of mutants determined by LRA

The newly determined LRA values are given in bold inTable 1. The basal cAMP activity measured by LRA us-

ing FACS analysis showed for M453T and L512Q an 4.5-5-fold increase compared to the wt followed by T632Iand D633Y which were 14.5-16-fold increased. I568T andF631L revealed with 25.6-fold and 45.9-fold, respective-ly, the highest increase in basal cAMP activity.The correlation of the clinical findings age at onset of HT,age at onset of goiter, and duration of HT with the LRAgave no significant results (p>0.05); the Spearman cor-relation coefficient was <0.8.The results of the test for association of the remainingclinical signs and findings with high or low LRA (Fisher’s-Exact-Test) are summarized in Table 2. The results of thenon-parametric tests for differences in LRA activity be-tweens groups of the clinical signs are shown in Table 3.No significant (exact significance, 2-tailed, p>0.05) asso-ciation of a clinical sign with a certain LRA group (high orlow LRA) was found. Moreover, no significant differencesin the median LRA values of the different categories ofthe clinical signs were observed.Iodine deficiency was described for 3 patients living inDenmark, France, and Italy (Table 1). As the follow-up ofthese patients was from 1996 until 2000, we referred todata from older iodine status investigations which de-scribed iodine deficiency for the respective countries atthe time of disease of the respective patients. Moreover,for the purpose of this investigation, we referred to uri-nary iodine data rather than goiter prevalence data. Fourpatients originating from Germany (1, 6, 7) and Bulgaria(14) were classified as iodine deficient at onset of HT. Io-dine deficiency was associated with 3 cases of fetal, 2

Table 1 - Functional characteristics of sporadic TSH receptor (TSHR) mutations.

TSHR mutation Fold basal in vitro cAMP SCA LRA Classification Iodine status of country of residency of(reference) production of TSHR mutation (reference) (reference) of LRA patient according to urinary iodine values

over WT TSHR1 (reference)

1. S281N (1) 3.5-fold (15) 6.8-fold (16) 6.0±2.6 (15) 15.1±2.9 (22) High (≥15.1) Mild iodine deficiency until 199611.9 (16) [Germany] (24)

2. S281N (2) 3.5-fold (15) 6.8-fold (16) 6.0±2.6 (15) 15.1±2.9 (22) High (≥15.1) Optimal [USA] (25)11.9 (16)

3. A428V (3) 6.4-fold (17) - 88.2±2.1 (17) High (≥15.1) Iodine sufficient [Patient was born in 2000][Germany] (24)

4. M453T (4) 7-fold - 5.2±0.8°2 Low (<15.1) Mild iodine deficiency [France] (26)

5. M453T (5) 7-fold - 5.2±0.8°2 Low (<15.1) Mild iodine deficiency [Denmark, Copenhagen,iodized salt not used until 1999] (26)

6. S505N (6) 5-fold 9.2±2.1(18) 4.5±0.3 (23) Low (<15.1) Mild iodine deficiency until 19966.5-fold (18) 3.7±0.5 (11) [Germany] (24)

7. S505N (7) 5-fold 9.2±2.1(18) 4.5±0.3 (23) Low (<15.1) Mild iodine deficiency until 19966.5-fold (18) 3.7±0.5 (11) [Germany] (24)

8. L512Q (8) 5-fold (19) - 4.5±0.7°2 Low (<15.1) Risk of iodine induced HT [Japan] (27)

9. I568T (9) 5.2-fold (20) - 25.6±6.3°2 High (≥15.1) Mild iodine deficiency [Italy] (26)

10. I568T (10) 5.2-fold (20) - 25.6±6.3°2 High (≥15.1) Optimal [USA] (25)

11. V597L (11) 2.4-fold 28.94±2.2 (11) 125.2±10.5 (22) High (≥15.1) Optimal [since 1998] [UK] (26)51.92 (21)

12. F631L (12) 5-6-fold - 45.9±9.4°2 High (≥15.1) Optimal [since 1980] [Switzerland] (28)

13. T632I (13) 3.7-fold (17) - 14.5±2.7°2 Low (<15.1) Optimal [USA] (25)

14. D633Y (14) 3-4-fold - 16.4±6.4°2 High (≥15.1) Mild iodine deficiency until 1996 [Bulgaria] (29)

WT: wild type; SCA: specific constitutive activity; LRA: linear regression analysis; HT: hyperthyroidism. LRA of basal activity as a function of TSHR expressiondetermined by 125I-bTSH binding or fluorescence-activated cell sorting analysis (°); WT was set at 1; values are expressed as means±SD of at least 3 in-dependent experiments, each performed in duplicate. Classification of LRA according to the different increases of LRA: high LRA>14.8; low LRA≤14.8.The iodine status in country of residency was determined according to urinary iodine values and not according to goiter prevalence data. 1( ) If report-ed in a further publication; 2newly determined LRA. Median urinary iodine in µg/l: optimal (100-199); mild deficiency (99-50); risk of iodine induced HT(200-299).

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230

cases of neonatal, and 2 cases of delayed onset of HT.The onset of HT in children originating from iodine-suffi-cient countries (UK, Switzerland, USA, Germany since1996) was 4 times during the neonatal period at the ageof 2 and 9 month. These data do not allow to draw anyclear-cut conclusions with regard to the relation of onsetof SNAH to the iodine status.

DISCUSSION

None of the 10 examined clinical signs showed a signif-icant correlation or association with a certain LRA value orwith one of the two LRA groups (high or low LRA). Thiswas most likely due to the restricted case number of 14patients and/or the presence of genetic/environmentalmodifiers. The inclusion of cases with familial non-au-toimmune HT caused by germline mutations (36-41) isnot possible since the CC of these families is differentfrom patients with SNAH (e.g. the onset of HT is usuallylater) and since there is considerable variability within thesame family and the CC is not reported in detail for allfamily members.Iodine supply is an obvious environmental modifier andtwo previous reports did suggest a relation between theiodine status and the onset of HT in two families withTSHR germline mutations (38, 42). Moreover, excessivethyroid enlargement was associated with iodine defi-ciency in 2 cases (7, 8). However, the available data onthe iodine status of the patients do not allow to draw anyclear-cut conclusions with regard to the relation of onset

of SNAH to the iodine status. In addition, the individualpatient’s situation might not be appropriately character-ized by these population data.No association of an active CC with a high LRA was ob-served. Therefore, other factors have to be taken into ac-count as shown by the case report of Nishihara et al. (8)who reported on a patient with excessive thyroid en-largement (370 ml) during 14 yr. This apparently activeCC is most likely primarily due to the insufficient treat-ment of HT in this patient due to a lack of compliance(which was supposed by the authors) and the known highiodine intake in Japan.The highest LRA was found for the patient described byEsapa et al. (11) (125.2±10.5) (Table 1). In comparison tothe other patients, this CC seems to be less complicatedas euthyroidism was rapidly achieved. However, in con-trast to other cases the decision for thyroid surgery wasmade rather early, after only 5 months of anti-thyroiddrug (ATD) therapy. Moreover, the published follow-upends at the time of the thyroid surgery and gives no in-formation of post-surgical relapses. This patient is, apartfrom the patient reported by Gruters et al. (1), the onlyone who underwent a total thyroidectomy as first surgery.Moreover, with regard to the remaining thyroid tissue theterm “total thyroidectomy” may have a different meaningin the different case reports.A very high LRA (88.2±2.1) was also described for the pa-tient reported by Borgel et al. (3). Neonatal HT was con-trolled with anti-thyroid treatment which was initially com-plicated by parental non-compliance. The patient was

Table 2 - Association of clinical signs of sporadic non-autoimmune hyperthyroidism (HT) with the linear regression analysis (LRA).

Clinical sign No. of patients with No. of patients with Frequency Frequency Exact significance, 2-tailed:low in vitro activity: high in vitro activity: of the of the Test for association

LRA≤14.8 LRA>14.8 clinical sign clinical sign of clinical parameters(Reference) (Reference) in patients with in patients with with high or low LRA(total no. 6) (total no. 8) LRA≤14.8 in % LRA>14.8 in % (Fisher’s-Exact-Test)

Onset of HT fetal 3 (4,5,6) 0 50 0 0.087neonatal 2 (8,13) 5 (2,3,9,10,12) 33.3 62.5late (>1 month) 1 (7) 3 (3,11,14) 16.7 37.5

Goiter age at ≤1 month 1 (4) 11 (12) 16.7 16.7 0.251onset of ≤15 months 1 (6) 41 (9-11,14) 16.7 66.7goiter >15 monts 4 (5,7,8,13) 11 (3) 66.7 16.7

morphology of goiter DG 4 (4,6,7,8) 51,2 (3,9,11,12,14) 66.7 100 0.455at onset of goiter MNG 2 (5,13) 0 33.3 0

excessive growth3 34 (6,7,8) 01 60 0 0.429

Prematurity 4 (4,5,8,13) 6 (1,2,9,10,12,14) 66.7 62.5 1

Mental retardation 4 (5,6,8,13) 2 (9,12) 66.7 33.3 0.567

Craniosynostosis 3 (6,8,13) 3 (2,3,14) 50 37.5 1

Low birth weight (<10th percentile) 3 (6,7,13) 1 (10) 50 12.5 0.245

Advanced bone age 5 (3-7) 6 (3,9-12,14) 83.3 75.0 1

Duration of HT (in yr) ≤1 04 4 (3,9,10,11) 0 50 0.2961<HT≤3 2 (6,7)4 1 (3) 40 16.7HT>3 3 (6,8,13)4 3 (1,12,14) 60 50

Active clinical course5 4 (5,6,8,13) 4 (1,10,12,14) 66.7 50 0.627

DG: diffuse goiter; MNG: multi-nodular goiter; HT: hyperthyroidism; LRA: linear regression analysis; ND: no data of follow-up; Neonatal: period from birth- 28 days of life. 1No goiter reported by Gruters et al., 1998 and Watkins et al, 2008. 2Morphology of goiter not reported by Borgel et al., 2005. 3Thedetailed follow-up of thyroid size in these 3 patients gives evidence for an excessive thyroid enlargement: Nishihara et al., 2006 reported a thyroid sizeof 370 ml at 20 yr; Holzapfel et al., 1997: 16 ml at 25 months, Fuhrer et al., 1999: 48 ml at 12.3 yr. 4No follow-up data for de Roux et al., 1996. 5Frequentrelapses of HT under anti-thyroid drug therapy, thyroid surgery and/or radio-iodine therapy plus complications of sporadic non-autoimmune HT such asprematurity, low birth weight, advanced bone age and mental retardation were characteristics of an active clinical course.

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231

clinically euthyroid for more than 2.6 yr, relapsed onlyonce, but euthyroidism was reinstalled by adjusting thedose of ATD therapy. Except from goiter she presentedno further complications. Therefore, we classified this CCas mild.Kopp et al. (12) reported on an active CC together witha high LRA (45.9±9.4). This patient was born premature-ly and presented goiter, advanced bone age, and men-tal retardation. As his relapsing HT was not controlled bya second surgery (excision of hyperplastic thyroid tissue)ablative radio-iodine therapy was applied. Afterwards heremained euthyroid. This patient certainly shows an activeCC, but it should be considered that this patient was notoperated until more than 8 yr of HT.These 3 cases illustrate the limitations of this systematicreview of case reports. The 2 patients with the highestIVA were classified as mild CC. However, this mild CCwas very likely due to the early appropriate treatment of

these patients and they might in fact be more appropri-ately classified as active CC thus instead supporting anassociation of high LRA with an active CC. Moreover, forsome patients, no detailed follow-up was reported andno data are available about further relapses of HT. Allthese circumstances can lead to misclassification of theCC. Finally, all patients were treated according to differ-ent therapeutic strategies which can change the CC andcan thus make comparisons difficult.For a more precise investigation of a relation betweengeno- and phenotype, different patients with the samesporadic germline mutation namely M453T, S505N,S281N, and I568T and consequently the same LRA werecompared.De Roux et al. (4) and Lavard et al. (5) reported 2 unre-lated patients with the TSH-receptor mutation M453T(Table 1). Both patients showed the fetal onset of HT andwere delivered before term. For the patient describedby de Roux et al. (4) euthyroidism could be easilyachieved with ATD.However, Lavard et al. (5), reported on a patient with se-vere HT. In early childhood, HT was controlled with ATD,but medical treatment and subtotal thyroidectomy couldnot stop thyroid hyperplasia and relapses of HT so thatrepeated ablative radio-iodine therapy (4 times from age9 to 13 yr, 131I doses of 150, 150, 200, and 500 MBq) wasnecessary. No compliance problems were reported.These 2 patients who both lived in countries with mildiodine deficiency (France and Denmark) and who had thesame LRA show a completely different CC.Moreover, S505N was described by Fuhrer et al. (7) andby Holzapfel et al. (6). Both patients were delivered atterm with low birth weight and showed excessive goitergrowth during several months/years. The patient’s CCreported by Holzapfel et al. (6) was severe as illustratedby fetal onset of disease, markedly advanced bone age,craniosynostosis, speech disturbances, and post-surgicalre-growth of the thyroid remnant tissue leading to a re-lapse of HT.Fuhrer et al. (7) described multiple relapses of HT as well,but the relapses were most likely caused by parental non-compliance (as reported by the authors). In spite of ex-cessive growth of the goiter under ATD the patient re-mained clinically euthyroid showing a milder course ofthe disease. Both patients grew up in Germany thus witha similar iodine supply.S281N was described in 2 patients (1, 2). The onset ofHT was neonatal and at 2 months. Gruters et al. (1) re-ported an active CC of a patient requiring surgery forcraniosynostosis and thyroid surgery for persistent HTwith relapse immediately after ATD dose reductionwhereas the patient described by Chester et al. (2)showed a milder CC, HT was controlled with ATD.A mild CC with neonatal onset of HT which was con-trolled with ATD associated with a I568T mutation wasreported by Tonacchera et al. (9), whereas Watkins et al.(10) reported a patient with I568T and a CC complicatedby meconium aspiration and pneumothrorax at birth andepisodic supraventricular tachycardia at the age of 9months.Therefore, the different CCs in these 8 patients also sug-gest that the activity of the CC is rather determined by

Clinical sign Median LRA Exact significance,value of 2-tailed:test forpatients differences ofwith the LRA-ranks (mann-

clinical sign Whitney-U-Testor Kruskal-

Wails-Test1)

Onset fetal 5.2 0.075-0.891of HT neonatal 25.6

late (>1 month) 15.8

Goiter age at ≤1 month 25.6 0.3-1.0onset of ≤15 months 16.4goiter >15 monts 5.2

morphology of goiter DG 15.1 0.745at onset of goiter MNG 9.85

excessive growth2 4.5 0.057

progressive growth 15.1

Prematurity 15.1 0.93Birth at term 46.35

Mental retardation 9.85 0.416No mental retardation 15.75

Craniosynostosis 14.8 0.175No craniosynostosis 25.6

Low birth weight (<10th percentile) 9.5 0.205Normal birth weight 15.75

Advanced bone age 15.1 0.599Normal bone age 15.1

Duration of HT (in yr) ≤1 25.6 0.175-0.7951<HT≤3 9.8HT>3 14.8

Active clinical course3 14.8 0.359Milder clinical course 20.35

DG: diffuse goiter; MNG: multi-nodular goiter; ND: no data of follow-up;Neonatal: period from birth - 28 days of life. 1Mann-Whitney-U-Test fortwo categories (e.g. prematurity: premature birth or birth at term) andKruskal-Walis-Test for 3 categories (e.g. onset of HT: fetal, neonatal orlate onset). 2The detailed follow-up of thyroid size in these 3 patientsgives evidence for an excessive thyroid enlargement: Nishihara et al.,2006 reported a thyroid size of 370 ml at 20 yr; Holzapfel et al., 1997:16 ml at 25 months, Fuhrer et al., 1999: 48 ml at 12.3 yr. 3 Frequent re-lapses of HT under anti-thyroid drug therapy, thyroid surgery and/or ra-dio-iodine therapy plus complications of sporadic non-autoimmune HTsuch as prematurity, low birth weight, advanced bone age, and mental re-tardation were characteristics of an active clinical course.

Table 3 - Median linear regression analysis (LRA) values of pa-tients clinical signs of hyperthyroidism (HT).

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232

further epigenetic/genetic, sex-related, and environ-mental factors than by the LRA of the mutated TSHR.Similar conclusions were recently reported for familialnon-autoimmune HT (38, 42).

CONCLUSION

None of the 10 examined clinical signs (namely age atonset of HT, age at onset of goiter, morphology of goi-ter, goiter growth, prematurity, mental retardation, cran-iosynostosis, low birth weight, advanced bone age, du-ration of HT) showed a significant correlation or associa-tion with the LRA values or with one of the two LRAgroups (high or low LRA).Considering the different diagnostic circumstances, ther-apeutic strategies and the limitations of a systematic anal-ysis of case reports due to limited follow-up and the re-stricted number of case reports, we found no clear evi-dence for a consistent relation of the TSHR mutation’sIVA determined by LRA with the CC of patients withSNAH. This may also be due to the action of genetic,epigenetic, and environmental modifiers.

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Shared Sporadic and Somatic Thyrotropin ReceptorMutations Display More Active In Vitro Activitiesthan Familial Thyrotropin Receptor Mutations

Julia Lueblinghoff,1 Markus Eszlinger,1 Holger Jaeschke,1 Sandra Mueller,1 Rifat Bircan,2

Hulya Gozu,3 Seda Sancak,4 Sema Akalin,4 and Ralf Paschke1

Background: Germline thyrotropin receptor (TSHR) mutations are associated with sporadic congenital non-autoimmune hyperthyroidism and familial nonautoimmune hyperthyroidism. Somatic TSHR mutations areassociated with toxic thyroid nodules (TTNs). The objective of the study was to define a relation of the clinicalappearance and the in vitro activity (IVA) of the TSHR mutations described by several authors for these thyroiddisorders.Methods: We analyzed the IVAs published as linear regression analysis (LRA) of the constitutive activity as afunction of the TSHR expression and the basal cyclic adenosine monophosphate (cAMP) values to determinedifferences between exclusively somatic, exclusively familial, and shared sporadic and somatic TSHR-mutations.Further, we investigated correlations of the LRAs/basal cAMP values with clinical activity characteristics(CACs) of TTNs, such as largest diameter of the TTN and the age of the patient at thyroid surgery.Results: Shared sporadic and somatic mutations showed higher median LRA (14.5) and higher median basalcAMP values (fivefold) than exclusively familial mutations (6.1, p¼ 0.0002; 2.9-fold, p< 0.0001, respectively).Moreover, mutations shared between sporadic congenital nonautoimmune hyperthyroidism and toxic thyroidnodules (TTNs) showed higher median LRA/basal cAMP values ( p< 0.0001) than exclusively somatic muta-tions in TTNs (5.1; 3.89-fold, respectively). Exclusively somatic mutations and exclusively familial mutationsshowed no significant difference in their median LRA values ( p¼ 0.786) but a significant difference for basalcAMP values ( p¼ 0.0006). The two examined CACs showed no correlation with the IVA characterized by LRA/basal cAMP values or with the presence or absence of a TSHR-mutation.Conclusions: This systematic analysis of published constitutively activating TSHR-mutations, their CACs, andtheir IVA provides evidence for higher IVA of shared sporadic and somatic TSHR mutations as compared withfamilial TSHR mutations. CACs of somatic TSHR mutations in TTNs did not have a clear association with theIVA as characterized by LRA or basal cAMP values.

Introduction

The phenomic characterization of cancers character-ized by specific mutations and molecular profiles, that is,

cancer phenomics has recently been introduced in the ‘‘omicsprofile.’’ Examples such as PTEN (protein-tyrosine phospha-tase) and RET (ret proto-oncogene) demonstrate that theevolution of phenomics will be crucial for the further ad-vancement of the understanding of oncogene signaling andpersonalized medicine also in the thyroid field (1–6).

Due to their slower growth, whichmost likely indicates lessactive oncogenes, the prominent role of environmental factorssuch as iodine and selenium, and also the relevance of severalintrathyroidal growth factors, the phenomic characteriza-tion of benign thyroid nodules seems to be more demandingas compared with cancers. Thus, in familial nonautoimmunehyperthyroidism (FNAH) within the same family with thesame germline mutation, the time of onset of hyperthyroid-ism (HT) can vary from 12 days to 36 years (7,8). For spo-radic congenital nonautoimmune hyperthyroidism (SCNAH),

1Division for Endocrinology and Nephrology, Department of Medicine, University of Leipzig, Leipzig, Germany.2Department of Molecular Biology, Faculty of Arts and Sciences, Namik Kemal University, Tekirdag, Turkey.3Section of Endocrinology and Metabolism, Dr. Lufti Kirdal Kartal Education and Research Hospital, Istanbul, Turkey.4Section of Endocrinology and Metabolism, Department of Internal Medicine, Marmara University Medical School, Istanbul, Turkey.

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there is no apparent correlation of the mutations’ in vitro ac-tivities (IVAs) with the clinical phenotype (5). Constitutivelyactivating germline thyrotropin receptor (TSHR) mutationshave been reported to date for 15 patients with sporadicmutations (http://innere.uniklinikum-leipzig.de/tsh; acces-sion date July 15, 1999) (9–23) and for 24 families with FNAH(http://innere.uniklinikum-leipzig.de/tsh, accession dateJuly 15, 1999) (7,24–47) (Table 1). A very preliminary analysisof 15 patients with hot thyroid nodules did not show signifi-cant differences in clinical presentation and thyroid hor-mone levels between patients with or without somatic TSHRmutations (48).

However, the consistent early onset of SCNAH has re-peatedly led to speculations whether those sporadic TSHRmutations are more active than the familial or somatic TSHRmutations. Based on a comparative gene expression analysis,a stronger effect of somatic TSHR mutations as comparedwith FNAH mutations has been recently suggested (49).However, a correlation analysis of clinical phenotypes within-vitro activities has not been performed to date and, exceptfor iodine-induced hyperthyroidism, the reasons for the largespectrum of markedly different clinical and in-vitro activi-ties and clinical courses of hot nodules (50) remain to be de-termined. Until now, 46 different point mutations and twodifferent deletions in the TSHR in TTNs (http://innere.uniklinikum-leipzig.de/tsh, accession date July 15, 1999)(51–76) (Table 1) have been described. Many of these TSHRmutations have been recently characterized for their IVA bylinear regression analysis (LRA) of constitutive activity as afunction of TSHR expression determined by 125I-bTSH bind-ing or FACS analysis compared with the wild-type TSHR,which is currently the most comprehensive in-vitro activitycharacterization method for TSHR mutations (5,77,78). We,therefore, used these data to investigate possible LRA differ-ences between exclusively somatic, exclusively familial, andshared sporadic and somatic TSHR mutations and to analyzethe correlation of LRAs with clinical activity characteristics ofhot thyroid nodules.

Materials and Methods

By a systematic analysis of the literature regarding allpublished constitutively activating TSHR mutations, weclassified and summarized the occurrence of the differentTSHR mutations as familial or sporadic germline (Table 1) oras somatic mutations (Table 1). Eleven different TSHR mu-tations were found in 15 cases of sporadic nonautoimmunehyperthyroidism (9–22). Seventeen different mutations werefound in 24 families with FNAH (7,24–46) and 46 somaticTSHR mutations, and two deletions were described for 310TTNs (51–74,79).

The LRA of constitutive activity as a function of TSHRexpression determined by 125I-bTSH binding or FACS anal-ysis compared with the wild-type TSHR was described as themost comprehensive way of characterizing the IVA of con-stitutively activating TSHR mutations (78,80–82). Therefore,we analyzed the TSHR mutations’ published LRAs by a sys-tematic review of the literature. As an example, the LRAsfor the TSHR mutations I568V and V597V are described inFigure 1.

As shown in Table 1 there are nine different exclusivelyfamilial TSHR mutations (described for 12 different families

with FNAH), 29 different exclusively somatic TSHR muta-tions (described for 116 different TTNs), and nine differentshared sporadic and somatic TSHRmutations (described in 11cases of SCNAH and 127 TTNs; all sporadic mutations canalso be found in TTNs). Therefore, we limited our groupcomparison to these three distinct groups of TSHRmutations.These three groups of TSHR mutations were tested for sig-nificant differences in the distribution of LRA values usinga nonparametric pairwise test (Mann-Whitney U-test). TheLRA values were available for four exclusively familial mu-tations (47,83) (described in 6 families), for 12 exclusively so-matic mutations (47,74,77,80,82–85) (described in 28 TTNs),and for eight shared sporadic and somatic TSHR mutations(5,80,83,84) (found in 10 patients with SCNAH and in 117TTNs).

Further, we compared the basal cyclic adenosine mono-phosphate (cAMP) values for significant differences betweenexclusively familial, exclusively somatic (one hot noduleat the time of surgery), and shared sporadic and somaticmutations (Mann-Whitney U-test). Basal cAMP values wereavailable for eight exclusively familial mutations (found in 11families), for 25 exclusively somatic mutations (found in 106TTNs), and for nine shared sporadic and somatic mutations(found in 11 patients with SCNAH and 117 TTNs). Sinceseveral basal cAMP values have been published for the samemutation and these values vary between laboratories, we in-cluded all the different values for the same TSHR mutation inthe pairwise testing.

To analyze the influence of a somatic TSHR mutation inTTNs on the patients’ phenotype, we retrospectively sys-tematically analyzed the clinical data of 75, 56, and 35 patientswith TTNs reported by Trulzsch et al. (76), Gozu et al. (84), andKrohn et al. (74), respectively. One hundred twenty-six of the166 patients reported in the three studies had single hotnodules (75 patients reported by Trulzsch et al., 36 patientsreported by Gozu et al., and 15 patients reported by Krohnet al.). Therefore, we investigated these 126 single hot nodulesfor a possible correlation of the largest diameter of the thyroidnodule with the LRA or with the basal cAMP value (dataavailable for 50 and for 134 patients, respectively) using abivariate correlation analysis (Spearman’s rho correlationcoefficient). Since different basal cAMP values have beenpublished for the same mutation and these values vary be-tween laboratories, we included all the different values for thesame TSHR mutation in the correlation analysis. Further, wetested the mean largest diameters of the single hot nodulesin patients with (n¼ 64) and without (n¼ 39) TSHR muta-tions for significant differences by t-test for two independentsamples.

The age of the 126 patients with single hot nodules at thetime of nodule surgery was investigated for a possible cor-relation with the LRA value (data available for 55 patients)and with the basal cAMP (data available for 152 patients)using a bivariate correlation analysis (Spearman’s rho corre-lation coefficient). Moreover, we compared the mean age ofpatients with single hot nodules with (n¼ 74) and without(n¼ 44) TSHR mutations for significant differences by thet-test for independent samples.

Statistical analysis was carried out with SPSS 15.0 forWindows and GraphPad Prism 5 for Windows Version11.0 (significant results: ***p< 0.001; **p¼ 0.001 to p< 0.01;*p¼ 0.01 to p< 0.05; p> 0.05 not significant).

222 LUEBLINGHOFF ET AL.

Table 1. Constitutively Activating Thyrotropin Receptor Mutations in Toxic Thyroid Nodules,

Sporadic Congenital Nonautoimmune Hyperthyroidism, and Familial Nonautoimmune Hyperthyroidism

Mutation number Somatic mutations in TTNs (refs.) Mutations in SCNAH (refs.) Mutations in FNAH (refs.)

1 M453T (54,56,63,72,73,76,84) M453T (11,18) M453T (45)2 S505N (76,84) S505N (13,15) S505N (43)3 S281N (53,55,63,71,73,74,76,84) S281N (14,21)4 A428V (73) A428V (10)5 L512Q (56,67,73,76,84) L512Q (19)6 I568T (55,56,62,63,73,76,84) I568T (20,22)7 V597L (73) V597L (12)8 I630L (57,58,63,70,73,84) I630L (23)9 F 631L (55,58,62,63,73,74,76,84) F 631L (16)10 T632I (52,54–58,63–66,68–71,73,74,76,84) T632I (17)11 D633Y (57,61,63,65,66,73,76,84) D633Y (9)12 G431S (73) G431S (26,44)13 M463V (73) M463V (25,29,31,35)14 V597F (73) V597F (24)15 A623V (52,55,57,61,64,66,70,73,74,76,84) A623V (40)16 L629F (56,57,57,63,70,71,73,74,84) L629F (30)17 P639S (70,84) P639S (34,70)18 A485V (7)19 S505R (32,38)20 V509A (33,41)21 I568V (27)22 D617Y (46)23 M626I (39,47)24 F631S (37)25 N650Y (42)26 C672Y (28)27 H271N (74)28 S281I (59)29 S281T (53,63,73,84)30 S425I (73,76)31 A428I (74,84)32 I486F (56,62,63,71,73,76,84)33 I486M (62,63,69–72,74)34 I486N (84)35 L512R (60,72,73,76,84)36 A593N (56)37 Y601N (51,56)38 D619G (55,61–63,66,68,70,73,76,84)39 A623C (74)40 A623F (73)41 A623I (62,63,73)42 A623S (52,66,86)43 A627V (84)44 F631C (65)45 F631I (74)46 T632A (71,72,74,76,84)47 D633A (63)48 D633E (55,56,63,70,71,73,74,76)49 D633H (57,63,66,73,74,76,84)50 I635V (73)51 P639A (76)52 I640K (84)53 A647V (71)54 V656F (55,73,76,84)55 F666L (68)56 Del 613–621 (55,76)57 Del 339–367 (75,79)

Mutations 1–27: Constitutively activating TSHR mutations found in TTNs, sporadic nonautoimmune hyperthyroidism (SCNAH), andFNAH. The references indicate the publications mentioning the detection of the respective TSHRmutation. Mutations 1–2: TSHRmutations inSCNAH, FNAH, and TTNs (two different mutations), mutations 3–11: TSHR mutations in SCNAH and TTNs (nine different mutations) (allsporadic mutations can also be found in TTNs), mutations 12–17: TSHR mutations in FNAH and somatic mutations in TTNs (six differentmutations),mutations 18–26: exclusively found in FNAH (nine differentmutations).Mutations 27–57: constitutively activating TSHRmutationsand deletions exclusively found in TTNs. The references indicate the publications mentioning the detection of the respective TSHR mutation.

FNAH, familial nonautoimmune hyperthyroidism; SCNAH, sporadic congenital nonautoimmune hyperthyroidism; TSHR, thyrotropinreceptor; TTN, toxic thyroid nodule.

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Results

Different distribution of LRA or basal cAMP values

for exclusively familial, exclusively somatic, and shared

sporadic and somatic TSHR mutations

Mutations shared between SCNAH and TTNs showedsignificantly higher median LRA (14.5) and basal cAMPvalues (fivefold) than mutations that were exclusively foundas familial mutations (6.1 with p¼ 0.0002; 2.9-fold withp< 0.0001, respectively) (Fig. 2a, b). Moreover, mutationsshared between SCNAH and TTNs showed significantly( p< 0.0001) higher median LRA/basal cAMP values thanmutations that were exclusively found as somatic mutationsin TTNs (5.1, 3.7-fold, respectively). Exclusively somatic mu-tations and exclusively familial mutations showed no sig-nificant difference for their median LRA values ( p¼ 0.786)(Fig. 2a), but there was a significant difference for basal cAMPvalues ( p¼ 0.0006, data mentioned above) (Fig. 2b).

Largest diameter of thyroid nodules

The single hot nodules showed no significant correlationof the largest diameter of the thyroid nodule with the LRAor with the basal cAMP value (r¼ 0.113, p¼ 0.436 with n¼ 50for the LRA values; r¼ 0.044, p¼ 0.613, n¼ 134 for the basalcAMP values). TSHR mutation positive and negative nod-ules showed mean largest diameters of 39.55 þ/ÿ13.25mm(n¼ 64) and 37.65 þ/ÿ13.97mm (n¼ 39), respectively( p¼ 0.537, no significant difference observed).

Age of the patients with hot nodules

A significant correlation of the age at thyroid surgerywith the LRA value (r¼ÿ0.269, p¼ 0.047, n¼ 55) was found

for the single hot nodules. No significant correlation of theage at thyroid surgery with the basal cAMP value (r¼ÿ0.125,p¼ 0.124, n¼ 152) was found. Mutation positive and negativenodules showed mean ages of 49.1 years (SD¼ 13.6, n¼ 74)and of 53.1 years (SD¼ 13.7, n¼ 44; p¼ 0.12, no significantdifference), respectively.

Discussion

Certain constitutively activating TSHR mutations and de-letions are exclusively found in TTNs, others are exclusivelyfound in FNAH (Table 1). However, all constitutively acti-vating TSHR mutations identified in children with SCNAHhave also been identified in TTNs; and two, in addition, havealso been found in FNAH. First indications for an exclusiveoccurrence of some TSHR mutations as FNAH mutationswere reported by Hebrant et al. (49). The analysis of furtherreports (9,10,19,36,52,53,55–58,60–63,65–68,70–73,76,84,86)indicates that there are currently 29 exclusively somatic mu-tations and nine exclusively familial mutations. Further, ninemutations are found as sporadic as well as somatic mutationsand two as sporadic as well as somatic mutations as well asfamilial mutations (Table 1). This identification of 29 exclu-sively somatic and nine exclusively familial constitutivelyactivating TSHR mutations by a systematic analysis of pub-lished reports including further in-vitro characterizationssubsequent to the original reports is the basis for our in-vitroactivity comparisons.

The exclusively familial mutations with published LRAvalues have lower LRA and basal cAMP activities than theshared sporadic and somatic mutations (Fig. 2a, b). Moreover,the shared somatic and sporadic mutations display a higherLRA and basal cAMP activity than the exclusively somaticmutations (Fig. 2a, b). Since the basal cAMP determinationswere done by different groups with slightly different meth-ods, the basal cAMP values are subject to broad variation.However, taken together these data also suggest that the highLRA activity of the shared sporadic and somatic mutations ismostly caused by the high IVA of the nine different sporadicmutations that are also found in the TTNs.

High LRA values in cases of familial germline mutationswill very likely lead to a survival disadvantage and, thus,would be less likely inherited by the next generation; whereasthe somatic mutations in TTNs occurring as de novo muta-tions are not affected by a similar negative genetic selection.On the contrary, to ensure its multiplication, the somaticde novo mutation requires a proliferation advantage (highIVA) compared with the other thyroid cells to manifest as ahot nodule (87). Moreover, somatic TSHR mutations occurlater in life whereas familial germline mutations are alreadypresent in all thyroid cells at the time of thyroid differentiationduring the embryonal period and start affecting the embryo atthat time. Thus, familial germline TSHR mutations affect thesurvival of the host much earlier than does the somatic mu-tation. Moreover, germline mutations are more likely to leadto hyperthyroidism, as they do not need to grow in compe-tition with normal thyroid tissue to become a tumor that islarge enough to cause hyperthyroidism as is the case of so-matic TSHR mutations. Taken together, somatic TSHR mu-tations are likely to be subject to positive selection; whereasfamilial germline TSHR-mutations are most likely subject tonegative selection. However, large phenotypic variability for

FIG. 1. Constitutive activity determined by linear regressionanalysis (LRA). Representative example of three slope deter-minations for mutations V597F and I568V (83) in comparisonto thewild-type (wt) thyrotropin receptor (TSHR). COS-7 cellsare transiently transfected with increasing amounts of DNA.Constitutive activity is expressed as basal cyclic adenosinemonophosphate (cAMP) formation (y-axis) as a function ofreceptor expression determined by FACS analysis (x-axis).Slopes were calculated using the linear regression function ofGraph Pad 4.0 for Windows. For comparison of the mutantsamong each other, the slope of the wt TSHR is set at 1; andslopes of the mutants are calculated accordingly.


the same familial TSHR mutation with ages of onset rang-ing from 12 days to 36 years (7) is a strong indication that thereare additional modifiers of TSHR mutation activity and/ordisease activity modifiers.

The dramatic clinical courses with first manifestation ofhyperthyroidism in utero (11,15,18) as well as excessive thy-roid enlargement, prematurity, craniosynostosis, mental re-tardation, and a markedly advanced bone age (13,15,19,59)accompanied by relapses of hyperthyroidism after thyroidsurgery (near-total or sub-total thyroidectomy) and/or afterradio-iodine therapy (9,15,16,18,23,59) described for patientswith SCNAH are in line with their distinctly higher basalcAMP activities. The higher IVA of the sporadic mutationscompared with the familial mutations (Fig. 2) is in line with

the earlier age of onset of hyperthyroidism in SCNAH than inFNAH. Ten of 15 patients with SCNAH had the onset of theirdisease during the first month of life and 5 of 15 during thefirst year of life, whereas only 4 of 24 patients with FNAH hadtheir onset during the neonatal period, specifically 3 of 24during the first year of life, and 17 of 24 until 21 years of life.Moreover, the LRA values of the shared sporadic and somaticmutations had the highest IVA, thus supporting the clinicalimpression of very active clinical courses of SCNAH as com-pared with the clinically mostly milder courses of patientswith TTNs. A recent functional and array data comparison ofthyroid tissues from FNAH and TTNs but not of SCNAHthyroid tissues showed similar but milder in vitro functionaland gene expression data for FNAH as compared with TTNs

FIG. 2. (a) Boxplot of the LRA values with median, 25%and 75% percentiles, minimum and maximum values of themutations found exclusively both as sporadic and somaticmutations, mutations exclusively found as familial muta-tions, and mutations found exclusively as somatic mutations(in toxic thyroid nodules [TTNs]). Gap at LRA value 30(exclusively familial mutations: minimum¼ 25% percentile,maximum¼ 75% percentile). The three groups of TSHRmutations were tested for significant differences in the dis-tribution of LRA values using a nonparametric pairwise test(Mann-Whitney U-test). There was no significant ( p¼ 0.786)difference between median LRA values of exclusively fa-milial and exclusively somatic mutations. Eight differentshared TSHR mutations in 10 cases of SCNAH and 117 TTNs(all sporadic mutations can also be found in TTNs: medi-an¼ 14.5, min¼ 4.5, max¼ 125.2 with n¼ 127. Four differentexclusively familial TSHR mutations in six different familieswith familial nonautoimmune hyperthyroidism: medi-an¼ 6.1, min¼ 1.9, max¼ 6.8, n¼ 6. Twelve different exclu-sively somatic TSHR mutations in 28 different TTNs:median¼ 5.1, min¼ 2.3, max¼ 25.1, n¼ 28. (b) Boxplot of thebasal cAMP fold values over wt (TSHR wt was set as 1) ofthe mutations found exclusively in both sporadic mutationsand TTNs, mutations exclusively found in familial muta-tions, and mutations exclusively found in somatic mutationsin TTNs. Gap at sevenfold basal cAMP value. The threegroups of TSHR mutations were tested for significant dif-ferences in the distribution of basal cAMP values using anonparametric pairwise test (Mann-Whitney U-test). Ninedifferent shared TSHR mutations in 11 cases of SCNAH and127 TTNs (more than one basal cAMP value was describedfor the eight different shared sporadic/somatic TSHR mu-tations, we included all values in our analysis, total numberof analyzed cAMP values¼ 294) (all sporadic mutations canalso be found in TTNs): median¼ 5.0-fold, min¼ 2.14-fold,max¼ 6.8-fold, n¼ 294. Eight different exclusively familialTSHR mutations in 12 different families with familial non-autoimmune hyperthyroidism (more than one basal cAMPvalue was described for the eight different TSHR mutations,we included all values in our analysis, total number of an-alyzed cAMP values¼ 24): median¼ 2.9-fold, min¼ 2.18-fold, max¼ sevenfold, n¼ 24. Twenty-five different exclu-sively somatic TSHR mutations in 106 different TTNs (morethan one basal cAMP value was described for the 25 differentexclusively TSHR mutations, we included all values in ouranalysis, total number of analyzed cAMP values¼ 227):median¼ 3.7-fold, min¼ 1.7-fold, max¼ 8.37-fold, n¼ 227.

DIFFERENT IVAS OF TSHR MUTATIONS 225

(49). Our analysis of other TSHR mutation in vitro dataof FNAH and TTNs support this conclusion. However, theelucidation of the possible pathophysiologic reasons for theseobservations require the additional analysis of SCNAH tis-sues, and our data together with correlating clinical data in-dicate that the more prominent IVAs of TSHR mutationsfound in SCNAH and TTNs as compared with FNAH couldbe a further important explanation.

The size of the hot thyroid nodule seems to be independentof the LRA value, the basal cAMP level, and the presenceor absence of a constitutively activating TSHR mutation. Ahigher LRA value or a higher basal cAMP is apparently notassociated with a younger age at surgery for patients with hotnodules, although there is aweak but significant correlation ofthe patient’s age at the time of surgery with the LRA value.Since only 7.3% of the variability of the patient’s age can beexplained by the different LRA values, 92.7% of the variabilityis influenced by other factors. No relation of the patient’s ageat thyroid surgery with or without the presence of a TSHRmutation was observed. It is obvious that the time of surgerywill depend onmany unrelated factors such as accessibility tosurgery, preference of the patient, or fear of malignancy.Moreover, the phenotype of a hot nodule is difficult to char-acterize, especially for the severity of hyperthyroidism, andsubject to several possible interferences and uncertaintiessuch as intracellular feedback mechanisms (88), further un-known genetic or epigenetic events, poor estimation of thetotal volume of active cells by the size of the adenoma, highinterobserver variation for thyroid nodule volume determi-nation (89), and differences in iodine supply. This could ex-plain the lack of genotype-phenotype correlations for hotnodules. Moreover, the constitutively activating TSHR mu-tations may not behave in human thyroid epithelial cellsin vivo in the same way as they do in transfected COS cells.This analysis was limited by the low numbers of cases in somegroups and by the fact that the groups for the comparison ofLRA and basal cAMPvalueswere not of equal size.Moreover,additional variants of other genes such as PDE 8B may in-fluence the net result of a TSHRmutation, and the result of thefunctional characterization of TSHR mutations may be dif-ferent in different cells (83).

Conclusion

This systematic analysis of published constitutively acti-vating TSHR mutations and their clinical and IVAs providesevidence for a higher IVA of shared sporadic and somaticTSHR mutations as compared with familial TSHR muta-tions. The few cases of sporadic nonautoimmune hyper-thyroidism for which very active clinical progression wasreported most likely contributed significantly to the higherLRA activity of the shared sporadic and somatic TSHRmutations. The two clinical indicators of the clinical activityof a somatic TSHR mutation that were examined, namelylargest diameter of the thyroid nodule and age of the patientat the time of thyroid surgery, showed no clear correlationwith the IVA characterized by the LRA value or the basalcAMP accumulation.

Acknowledgments

We thank H. Berger (Institute of Medical Statistics, Uni-versity of Leipzig, Leipzig, Germany) for help with the sta-

tistical analysis. This research was funded by DFG grants Pa423/14-1, Pa 423/15-2, and and Ja 927/1-1.

Disclosure Statement

The authors have nothing to declare. No competing fi-nancial interests exist.

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70. Tonacchera M, Chiovato L, Pinchera A, Agretti P, Fiore E,Cetani F, Rocchi R, Viacava P, Miccoli P, Vitti P 1998Hyperfunctioning thyroid nodules in toxic multinodulargoiter share activating thyrotropin receptor mutations withsolitary toxic adenoma. J Clin Endocrinol Metab 83:492–498.

71. Tonacchera M, Agretti P, Chiovato L, Rosellini V, CeccariniG, Perri A, Viacava P, Naccarato AG, Miccoli P, Pinchera A,Vitti P 2000 Activating thyrotropin receptor mutations arepresent in nonadenomatous hyperfunctioning nodules oftoxic or autonomous multinodular goiter. J Clin EndocrinolMetab 85:2270–2274.

72. Vanvooren V, Uchino S, Duprez L, Costa MJ, Vandekerc-khove J, Parma J, Vassart G, Dumont JE, Van SJ, Noguchi S2002 Oncogenic mutations in the thyrotropin receptor ofautonomously functioning thyroid nodules in the Japanesepopulation. Eur J Endocrinol 147:287–291.

73. Palos-Paz F, Perez-Guerra O, Cameselle-Teijeiro J, Rueda-Chimeno C, Barreiro-Morandeira F, Lado-Abeal J, AraujoVD, Argueso R, Barca O, Botana M, Cabezas-Agricola JM,Catalina P, Dominguez GL, Fernandez T, Mato A, Nuno A,Penin M, Victoria B, Paloz-Paz F, Perrez-Guerra O, Came-selle-Teijeiro J, Rueda-Chimio JC, Barreio-Morandeira F 2008Prevalence of mutations in TSHR, GNAS, PRKAR1A andRAS genes in a large series of toxic thyroid adenomas fromGalicia, an iodine-deficient area in NW Spain. Eur J Endo-crinol 159:623–631.

74. Krohn K, Sancak S, Jaeschke H, Semiha Sen L, GulluogluBM, Tanav Eran F, Sever Z, Sema Akalin N, Paschke R.,Eszlinger M 2008 High prevalence of TSHR/Gsa-mutation-negative clonal toxic thyroid nodules in a turkish cohort.Hormones Suppl 1:42.

75. Kosugi S, Akamizu T, Takai O, Prabhakar BS, Kohn LD1991 The extracellular domain of the TSH receptor hasan immunogenic epitope reactive with Graves’ IgG butunrelated to receptor function as well as determinants havingdifferent roles for high affinity TSH binding and the activityof thyroid-stimulating autoantibodies. Thyroid 1:321–330.

76. Trulzsch B, Krohn K, Wonerow P, Chey S, Holzapfel HP,Ackermann F, Fuhrer D, Paschke R 2001 Detection of thy-roid-stimulating hormone receptor and Gsalpha mutations:in 75 toxic thyroid nodules by denaturing gradient gelelectrophoresis. J Mol Med 78:684–691.

77. Jaeschke H, Kleinau G, Sontheimer J, Mueller S, Krause G,Paschke R 2008 Preferences of transmembrane helices forcooperative amplification of G(alpha)s and G (alpha)q sig-naling of the thyrotropin receptor. Cell Mol Life Sci 65:4028–4038.

78. Gozu HI, Mueller S, Bircan R, Krohn K, Ekinci G, Yavuzer D,Sargin H, Sargin M, Ones T, Gezen C, Orbay E, Cirakoglu B,Paschke R 2008 A new silent germline mutation of the TSH

receptor: coexpression in a hyperthyroid familymember witha second activating somatic mutation. Thyroid 18:499–508.

79. Zhang ML, Sugawa H, Kosugi S, Mori T 1995 Constitutiveactivation of the thyrotropin receptor by deletion of a por-tion of the extracellular domain. Biochem Biophys ResComm 211:205–210.

80. Jaeschke H, Neumann S, Kleinau G, Mueller S, Claus M,Krause G, Paschke R 2006 An aromatic environment in thevicinity of serine 281 is a structural requirement for thyro-tropin receptor function. Endocrinology 147:1753–1760.

81. Vlaeminck-Guillem V, Ho SC, Rodien P, Vassart G,Costagliola S 2002 Activation of the cAMP pathway by theTSH receptor involves switching of the ectodomain from atethered inverse agonist to an agonist. Mol Endocrinol 16:736–746.

82. Kleinau G, Jaeschke H, Mueller S, Raaka BM, Neumann S,Paschke R, Krause G 2008 Evidence for cooperative signaltriggering at the extracellular loops of the TSH receptor.FASEB J 22:2798–2808.

83. Mueller S, Gozu HI, Bircan R, Jaeschke H, Eszlinger M,Lueblinghoff J, Krohn K, Paschke R 2009 Cases of borderlinein vitro constitutive thyrotropin receptor activity: how todecide whether a thyrotropin receptor mutation is consti-tutively active or not? Thyroid 19:765–773.

84. Gozu HI, Bircan R, Krohn K, Muller S, Vural S, Gezen C,Sargin H, Yavuzer D, Sargin M, Cirakoglu B, Paschke R 2006Similar prevalence of somatic TSH receptor and Gsalphamutations in toxic thyroid nodules in geographical regionswith different iodine supply in Turkey. Eur J Endocrinol155:535–545.

85. Kleinau G, Jaeschke H, Mueller S, Worth CL, Paschke R,Krause G 2008 Molecular and structural effects of inverseagonistic mutations on signaling of the thyrotropin recep-tor—a basally active GPCR. Cell Mol Life Sci 65:3664–3676.

86. Russo D, Arturi F, Suarez HG, Schlumberger M, du VillardJA, Crocetti U, Filetti S 1996 Thyrotropin receptor genealterations in thyroid hyperfunctioning adenomas. J ClinEndocrinol Metab 81:1548–1551.

87. Krohn K, Fuhrer D, Bayer Y, Eszlinger M, Brauer V, Neu-mann S, Paschke R 2005 Molecular pathogenesis of euthy-roid and toxic multinodular goiter. Endocr Rev 26:504–524.

88. Kursawe R, Paschke R 2007 Modulation of TSHR signal-ing by posttranslational modifications. Trends EndocrinolMetab 18:199–207.

89. Brauer VF, Eder P, Miehle K, Wiesner TD, Hasenclever H,Paschke R 2005 Interobserver variation for ultrasound deter-mination of thyroid nodule volumes. Thyroid 15:1169–1175.

Address correspondence to:Ralf Paschke, M.D.

Division for Endocrinology and NephrologyDepartment of Medicine

University of LeipzigPhilipp Rosenthal St. 27

Liebigstraße 20; Leipzig 04103Germany

E-mail: [email protected]


27

V. Zusammenfassung

Dissertation zur Erlangung des akademischen Grades

Dr. med.

Klinische Charakterisierung von TSH-Rezeptormutatio nen

eingereicht von

Julia Cordula Lüblinghoff

angefertigt am

Universitätsklinikum Leipzig

Medizinische Klinik und Poliklinik für Endokrinologie und Nephrologie

Liebigstraße 20, D – 04103 Leipzig

betreut von

Prof. Dr. med. R. Paschke

eingereicht im November 2011

Klinische Charakterisierung sporadischer TSH-Rezept ormutationen

Der erste Teil dieser kumulativen Dissertation (S. 12-17) konzentriert sich auf die klinische

Charakterisierung der sporadischen TSH-Rezeptormutationen. Die 14 bis dato beschrie-

benen Patienten6 wurden bezüglich ihrer klinischen Merkmale untersucht (z.B. Frühge-

burtlichkeit, geringes Geburtsgewicht, Alter bei Erstmanifestation der Hyperthyreose, vor-

zeitige Knochenreifung, Schilddrüsenvolumen und -wachstum). Anschließend wurden die

14 Patienten gemäß der Linearen Regressions-Analyse (LRA) in zwei Gruppen eingeteilt

(Patienten mit hohem LRA-Wert, LRA ≥ 15.1 und Patienten mit niedrigem LRA-Wert,

LRA < 15.1; der Zahlenwert 15.1 wurde als Kriterium gewählt, da er dem Median der LRA-

Werte aller sporadischen Mutationen entspricht) (Anlagen: Tabelle A, S. 45). Die Häufig-

keit des Auftretens der o.g. Merkmale wurde mit Hilfe des Fisher’s Exakt-Test auf signifi-

6 Zwei Patienten (I630L, Bertalan et al., 2010 (6) und I486N, Biebermann et al., 2011 (8)) wurden erst nach Veröffentlichung des ersten Artikels dieser kumulativen Dissertation beschrieben. Eine Neuberechnung zeigte keinen signifikanten Unterschied zu den publizierten Ergebnissen.

28

kante Unterschiede zwischen den zwei Gruppen getestet. Außerdem erfolgte ein Ver-

gleich der LRA-Mediane für bestimmte Merkmalsgruppen (z.B. Alter bei Erstmanifestation

der Hyperthyreose: in der Fetalperiode, in der Neonatalperiode, nach dem ersten Le-

bensmonat; Frühgeburtlichkeit verglichen mit termingerechter Geburt etc.). Für vier Muta-

tionen wurden jeweils zwei Patienten beschrieben, deren klinische Verläufe direkt mitein-

ander verglichen wurden (Anlagen: Tabelle A). Außerdem erfolgte eine Analyse des Phä-

notyps unter Berücksichtigung der Jodversorgung des Patienten.

Keines der zehn untersuchten klinischen Merkmale zeigte eine Assoziation mit der LRA-

Aktivität der sporadischen TSH-Rezeptormutation. Auch der Vergleich der Patienten mit

der gleichen sporadischen TSH-Rezeptormutation zeigte keinen Zusammenhang zwi-

schen der in vitro Aktivität und dem beschriebenen Krankheitsverlauf. Die Jodversorgung

des Patienten scheint keinen Einfluss auf den klinischen Verlauf zu haben, wie sich z.B.

am Alter der Erstmanifestation und des Schilddrüsenvolumens zeigte.

Vergleich der in vitro Aktivität (LRA- und cAMP-Wer te) aller beschriebenen

konstitutiv aktivierenden TSH-Rezeptormutationen

Im nächsten Arbeitsschritt (S. 18-26) erfolgte ein Vergleich der in vitro Aktivität (LRA- und

cAMP-Werte) aller beschriebenen konstitutiv aktivierenden TSH-Rezeptormutationen.

Auffällig war, dass einige der Mutationen sowohl als sporadische, familiäre und somati-

sche Mutationen auftreten, andere hingegen ausschließlich bei den familiären bzw. den

somatischen Mutationen (Anlagen: Tabelle 1.1 und 1.2., S. 41, 42). Alle sporadischen Mu-

tationen finden sich auch bei den autonomen Adenomen wieder. Es erfolgte ein Vergleich

der in vitro Aktivitäten (LRA- und cAMP-Werte) in drei verschiedenen Gruppen: aus-

schließlich familiäre bzw. ausschließlich somatische Mutationen und gemeinsame spora-

dische und somatische TSH-Rezeptormutationen (Abbildung A, nächste Seite). Hierbei

zeigte sich eine signifikant höhere in vitro Aktivität der gemeinsamen sporadischen und

somatischen Mutationen verglichen mit den ausschließlich familiären und den ausschließ-

lich somatischen Mutationen. Dieses Ergebnis entspricht dem beschriebenen Krankheits-

verlauf der sporadischen nicht-autoimmunen Hyperthyreose (SNAH). Für die wenigen be-

schriebenen Fälle der SNAH wurden schwerwiegendere Verläufe berichtet als für die fa-

miliären nicht-autoimmunen Hyperthyreosen und die autonomen Adenome.

Für die Untersuchung der autonomen Adenome (AA) wurden Daten über klinische Merk-

male verwendet, welche von Trülzsch et al. 2001 (89), Gözu et al. 2006 (29) und Sancak

et al. 2011 (76) erhoben wurden. Diese drei Autoren beschrieben Patienten-Kollektive mit

AA, bei denen mit unterschiedlicher Häufigkeit somatische TSH-Rezeptormutationen

29

nachgewiesen wurden (75%, 70.2% und 40%, respektive). Es erfolgte eine Korrelation

des Patientenalters zum Zeitpunkt der Schilddrüsenoperation und des Knotenvolumens

mit den entsprechenden LRA- bzw. cAMP-Werten. Außerdem wurden die Mittelwerte des

Patientenalters zum Zeitpunkt der Schilddrüsenoperation und des Knotenvolumens bei

Patienten mit bzw. ohne TSH-Rezeptormutationen auf signifikante Unterschiede getestet.

Es zeigte sich kein eindeutiger Zusammenhang zwischen Knotenvolumen bzw. dem Alter

der Patienten und der entsprechenden in vitro Aktivität oder mit dem Vorhandensein bzw.

der Abwesenheit einer TSH-Rezeptormutation.

spor

adisc

h und

somat

isch

auss

chli eß

lich f

ami liä

r

auss

chl ie

ßlich so

mat

isch

0

10

20

30

80130

p = 0.0002p < 0.0001

n = 127 n = 28n = 6

LRA

-Wer

t

Abbildung A: Boxplot der LRA-Werte mit

Median, 25 und 75%-Perzentilen sowie

Minimum und Maximum für gemeinsame

sporadische und somatische Mutationen,

ausschließlich familiäre Mutationen und

ausschließlich somatische Mutationen.

Unterbrechung der Skalierung bei LRA-

Wert 30. Die 3 Gruppen wurden auf signi-

fikante Unterschiede in der Verteilung der

LRA-Werte getestet (Mann-Whitney-U-

Test). Kein signifikanter Unterschied zwi-

schen ausschließlich familiären und aus-

schließlich somatischen Mutationen.

Schlussfolgerung

Unter Berücksichtigung der kleinen Fallzahl und des kurzen Beobachtungszeitraumes der

sporadischen nicht-autoimmunen Hyperthyreose und den unterschiedlichen Diagnose-

und Therapiestrategien konnte kein eindeutiger Zusammenhang zwischen dem Phänotyp

der sporadischen TSH-Rezeptormutationen und der gemessenen LRA-Aktivität oder der

Jodversorgung des Patienten nachgewiesen werden. Für die gleiche Mutation wurden un-

terschiedliche Krankheitsverläufe beschrieben. Der klinische Verlauf scheint von weiteren

epigenetischen, genetischen und geschlechtsspezifischen Faktoren, der Behandlungs-

strategie und Umweltfaktoren abhängig zu sein. Die Relevanz einer adäquaten Therapie

zeigt sich z.B. bei Patienten mit früher totaler Thyroidektomie und nachfolgender anhal-

tender Euthyreose (20) im Gegensatz zur medikamentösen Therapie, für die zahlreiche

Rückfälle der Erkrankung berichtet wurden (30; 50). Die Malcompliance eines Patienten

30

ist ebenfalls ein wichtiger Faktor, welcher den klinischen Verlauf maßgeblich beeinflusst

(61).

Der Vergleich der in vitro Aktivitäten der ausschließlich familiären, ausschließlich somati-

schen und gemeinsamen sporadischen und somatischen Mutationen zeigte eine höhere

in vitro Aktivität der zehn gemeinsamen sporadischen und somatischen Mutationen im

Verhältnis zu den ausschließlich familiären und den ausschließlich somatischen Mutatio-

nen. Alle beschriebenen sporadischen Mutationen lassen sich auch bei den somatischen

Mutationen der AA nachweisen. Der Einfluss der sporadischen Mutationen scheint die

deutlich höhere in vitro Aktivität der gemeinsamen sporadischen und somatischen Mutati-

onen im Vergleich zu den ausschließlich familiären zu bewirken. Dies entspricht auch den

milden Krankheitsverläufen, welche für die familiären Mutationen berichtet wurden. Ein

hoher LRA-Wert würde bei einer familiären Keimbahnmutation zu einem Überlebensnach-

teil führen und somit mit geringerer Wahrscheinlichkeit vererbt werden, wohingegen die

somatischen Mutationen in den AA als de novo Mutationen auftreten und nicht von einer

negativen Selektion betroffen sind. Andererseits benötigt die somatische de novo Mutati-

on einen Proliferationsvorteil (die hohe in vitro Aktivität) im Vergleich zum umgebenden

Schilddrüsengewebe, um ihre Vervielfältigung zu ermöglichen und einen heißen Knoten

bilden zu können (49). Die Manifestation einer somatischen Mutation erfolgt zu einem

späteren Zeitpunkt, während die familiären Keimbahnmutationen bereits in allen Schild-

drüsenzellen in der Embryonalperiode in der Phase der Gewebedifferenzierung vorliegen.

Damit beeinflusst die familiäre Mutation das Überleben des Mutationsträgers schon zu ei-

nem früheren Zeitpunkt als die somatische Mutation. Zusammenfassend lässt sich sagen,

dass die somatischen TSH-Rezeptormutationen eher von einer Positivselektion betroffen

sind, die familiären Keimbahnmutationen eher von einer Negativselektion. Trotzdem wei-

sen die variierenden Phänotypen der familiären nicht-autoimmunen Hyperthyreose

(FNAH) mit einem Erstmanifestationsalter von 12 Tagen bis zu 36 Jahren bei der gleichen

Mutation innerhalb einer Familie (1) auf weitere Einflussfaktoren auf den Krankheitsverlauf

hin.

Die dramatischen klinischen Verläufe mit Erstmanifestation der Hyperthyreose in utero

(14; 33; 50), exzessiver Schilddrüsenvergrößerung, Frühgeburtlichkeit, Kraniosynostose,

mentaler Retardierung und vorzeitiger Knochenreifung (24; 33; 44; 61) zusammen mit

zahlreichen Rückfallen der Hyperthyreose nach operativer Therapie (subtotaler Thyreoi-

dektomie) und / oder Radiojodtherapie (6; 9; 33; 44; 45; 50), welche für Patienten mit

SNAH beschrieben wurden, stimmen mit den hohen LRA-Werten der sporadischen Muta-

tionen überein. Die höhere in vitro Aktivität der sporadischen Mutationen im Vergleich zu

den familiären Mutationen passt zur späteren Erstmanifestation der FNAH. Zehn der 15

Patienten mit einer SNAH zeigten erste Symptome im ersten Lebensmonat, bei den

31

FNAH waren es nur fünf von 90. Die gemeinsamen sporadischen und somatischen Muta-

tionen hatten die höchsten LRA-Werte. Dies entspricht auch dem klinischen Eindruck von

sehr aktiven Verläufen der SNAH im Vergleich zu relativ milden Verläufen der Patienten

mit FNAH. Eine kürzlich publizierte Studie verglich funktionale Eigenschaften der AA und

der FNAH und zeigte ähnliche aber mildere in vitro Aktivitäts- und Gen-Expressionswerte

der FNAH im Vergleich zu den AA (31). Dies entspricht auch unseren Ergebnissen beim

Vergleich der in vitro Aktivitäten der FNAH und der AA. Um die pathophysiologischen Ur-

sachen näher zu beleuchten, hätte man zusätzlich noch das Schilddrüsengewebe der

SNAH untersuchen können. Trotzdem zeigen unsere Daten im Zusammenhang mit dem

klinischen Verlauf einen stärkeren Einfluss der in vitro Aktivität der SNAH und der AA im

Vergleich zu den FNAH.

Die Größe eines heißen Schilddrüsenknotens scheint von der in vitro Aktivität und der An-

bzw. der Abwesenheit einer TSH-Rezeptormutation unabhängig zu sein. Eine hohe in

vitro Aktivität zeigt keinen Zusammenhang mit einem jüngeren Patientenalter zum Zeit-

punkt der Operation, obwohl eine schwache aber signifikante positive Korrelation zwi-

schen dem Patientenalter und den LRA-Werten beobachtet wurde. Allerdings lassen sich

nur 7,3% der Variabilität des Patientenalters mit den LRA-Werten erklären. Es zeigte sich

außerdem kein Zusammenhang zwischen Patientenalter und der Ab- bzw. Anwesenheit

einer TSH-Rezeptormutation. Der Zeitpunkt der operativen Entfernung eines autonomen

Adenoms und damit das Alter des Patienten zum Zeitpunkt der Operation ist von mehre-

ren Faktoren abhängig wie z.B. der Verfügbarkeit einer operativen Therapie, den Präfe-

renzen des Patienten bzw. der Angst vor einer malignen Erkrankung und ist somit starken

Schwankungen unterworfen. Unter Berücksichtigung der unterschiedlich großen Fallzah-

len der untersuchten Gruppen, der Untersucher-abhängigen Varianz der Größenbestim-

mung der heißen Knoten und der unterschiedlichen Jodversorgung der Patienten ließ sich

keine eindeutige Korrelation des Phänotyps mit dem Genotyp der AA feststellen. Außer-

dem verhalten sich die TSH-Rezeptormutationen in den Schilddrüsenzellen anders als in

den COS-7 Zellen, welche für die Messung der in vitro Aktivität verwendeten wurden.

Unsere Untersuchungen zeigten eine höhere in vitro Aktivität der sporadischen Mutatio-

nen im Vergleich zu den familiären und den somatischen Mutationen, auch wenn die Kor-

relation klinischer Merkmale der SNAH mit den in vitro Aktivitäten keinen direkten Zu-

sammenhang ergab. Die hohe in vitro Aktivität der sporadischen Mutationen stimmt mit

den dramatischen klinischen Verläufen überein, welche für diese Erkrankung beschrieben

worden sind.

32

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34

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VII. Anlagen

Tabelle 1.1 und 1.2: Konstitutiv aktivierende TSH-Rezeptormutationen in autonomen

Adenomen, sporadischer nicht-autoimmuner Hyperthyreose und familiärer nicht-

autoimmuner Hyperthyreose. Stand Oktober 2011

Abbildung 2: Konstitutiv aktivierende Mutationen des TSH-Rezeptors

Tabelle A: Funktionale Eigenschaften sporadischer TSH-Rezeptormutationen

41

Tabelle 1.1

Tabelle 1.1 (Mutationen 1-29) (Fortsetzung nächste Seite): Konstitutiv aktivierende TSH-

Rezeptormutationen in autonomen Adenomen (AA), sporadischer nicht-autoimmuner Hyperthyreo-

se (SNAH) und familiärer nicht-autoimmuner Hyperthyreose (FNAH).

Die Quellenangaben beziehen sich auf die Publikationen, welche die entsprechende

TSH-Rezeptormutationen identifiziert haben. Stand Oktober 2011

7 N670S und I691F sind nicht konstitutiv aktiv vgl. (38)

Nr der Mutation

Somatische Mutationen in AA Mutationen bei SNAH Mut ationen bei FNAH

1 M453T (16; 27; 29; 48; 58; 65; 67; 89; 92)

M453T (14; 50) M453T (79)

2 S505N (29; 58; 89) S505N(24; 33) S505N (91) 3 S281N (17; 23; 29; 58; 65; 67; 76;

84; 89) S281N (12; 30)

4 A428V (65) A428V (10) 5 I486N (29) I486N (8) 6 L512Q (27; 29; 65; 80; 89) L512Q (61) 7 I568T (23; 27; 29; 58; 65; 67; 68; 89) I568T (85; 94) 8 V597L(65) V597L (20) 9 I630L (28; 29; 32; 58; 65; 67; 86) I630L (6) 10 F631L (23; 29; 32; 58; 65; 67; 68;

76; 89) F631L (45)

11 T632I (15; 16; 23; 27-29; 32; 58; 65; 67; 70; 72; 75; 76; 82; 84; 86; 88; 89)

T632I (43)

12 D633Y (28; 29; 63; 65; 67; 72; 75; 89)

D633Y (9)

13 G431S (65) G431S (7; 19) 14 M463V (65) M463V (4; 21; 25; 51) 15 V597F (65) V597F (2) 16 A623V (15; 23; 28; 29; 63; 65; 70;

75; 76; 86; 89) A623V (78)

17 L629F (27; 28; 28; 29; 65; 67; 76; 84; 86)

L629F (26)

18 P639S (29; 86) P639S (42; 86) 19 A485V (1) 20 S505R (34; 71) 21 V509A (41; 83) 22 I568V (13) 23 E575K (59) 24 D617Y (62) 25 M626I (36; 73) 26 F631S (64) 27 C636W (95) 28 N650Y (87) N670S (87)7

29 C672Y (18) I691F (52)7

42

Tabelle 1.2

Tabelle 1.2 (Mutationen 30 – 60) (Fortsetzung von Tabelle 1.1): Konstitutiv aktivierende TSH-

Rezeptormutationen in autonomen Adenomen (AA), sporadischer nicht-autoimmuner Hyperthyreo-

se (SNAH) und familiärer nicht-autoimmuner Hyperthyreose (FNAH)

Die Quellenangaben beziehen sich auf die Publikationen, welche die entsprechende

TSH-Rezeptormutationen identifiziert haben. Stand September 2011

8 F666L ist nicht konstitutiv aktiv vgl. (38)

Nr der Mutation

Somatische Mutationen in AA

30 H271N (76) 31 S281I (44) 32 S281T (17; 29; 65; 67) 33 S425I (65; 89) 34 A428I (29; 76) 35 I486F (27; 29; 65; 67; 68; 84; 89) 36 I486M (58; 67; 68; 76; 84; 86; 88; 92) 37 L512R (29; 47; 65; 89; 92) 38 A593N (27) 39 Y601N (3; 27) 40 D619G (23; 29; 63; 65; 67; 68; 75; 82; 86; 89) 41 A623C (76) 42 A623F (65) 43 A623I (65; 67; 68) 44 A623S (15; 74; 75) 45 A627V (29) 46 F631C (72) 47 F631I (58; 76) 48 T632A (29; 76; 84; 89; 92) 49 D633A (67) 50 D633E (23; 27; 58; 65; 67; 76; 84; 86; 89) 51 D633H (28; 29; 58; 65; 67; 75; 76; 89) 52 I635V (65) 53 P639A (89) 54 I640K (29) 55 A647V (84) 56 V656F (23; 29; 65; 89) F666L (82)8 57 Del D403 (60) 58 Del Y613-621 (23; 89) 59 Del E339-367 (46; 96)

43

Abbildung 2

Abbildung 2: Konstitutiv aktivierende Mutationen des TSH-Rezeptors

Dargestellt ist der TSH-Rezeptor mit N-Terminus, Extrazellulardomäne, den sieben Trans-

membran Domänen, je drei intra- und extrazellulären Schleifen, der Intrazellulardomäne

und der C-Terminus. Die 764 Aminosäuren sind entsprechend der Ballesteros-Weinstein

Nomenklatur bezeichnet (5). Die Abbildung zeigt zwölf sporadische TSH-

Rezeptormutationen (blaue Raute), 19 familiäre Mutationen (rotes Quadrat), 45 somati-

sche Mutationen (gelber Kreis) und 3 konstitutiv aktivierenden Deletionen (* bzw **). Kur-

siv gedruckt sind Mutationen ohne konstitutive Aktivität (I691F, F666L und N670S)

*D

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45

Tabelle A

Erläuterungen:

LRA = Die Lineare Regressions-Analyse der basalen Rezeptoraktivität als Funktinon der TSH-

Rezeptor-Expression (mittels FACS Analyse (Fluorescence Activated Cell Sorting) oder mittels

Bindung des radioaktiv markierten 125I-bTSH (bovine TSH) quantifiziert). Der Wildtyp TSH-Rezeptor

wird mit 1 gleich gesetzt. Die Werte sind Mittelwerte +/- Standardabweichung von mindestens drei

unabhängigen Experimenten, welche als Duplikat durchgeführt wurden.

Fettgedruckt sind LRA-Werte, die im Rahmen dieser Dissertation neu bestimmt wurden.

Zu 5. Biebermann et al., 2011 und zu 16. Bertalan et al., 2010: Diese Mutationen wurde erst nach

Veröffentlichung der ersten Publikation dieser Dissertation berichtet.

Tabelle A: Funktionale Eigenschaften sporadischer TSH-Rezeptormutationen

(modifiziert (53))

TSH-Rezeptormutation (Quellenangabe)

LRA (Quellenangabe)

Einteilung der LRA-Werte entsprechend dem

Median-LRA-Wert aller sporadischen Mutationen

1. S281N (30) (Grüters et al., 1998) 15.1±2.9 (55). hoch (≥15.1)

2. S281N (12) (Chester et al., 2008) 15.1±2.9 (55) hoch (≥15.1)

3. A428V (10) (Börgel et al., 2005) 88.2±2.1 (29) hoch (≥15.1)

4. M453T (14) (de Roux et al., 1996) 5.2±0.8 niedrig (<15.1)

5. M453T (50) (Lavard et al., 1999) 5.2±0.8 niedrig (<15.1)

6. I486N (8) (Biebermann et al., 2011) 6.7±1.4 (29) niedrig (<15.1)

7. S505N (33) (Holzapfel et al., 1997) 4.5±0.3 (37) niedrig (<15.1)

8. S505N (24) (Führer et al., 1999) 4.5±0.3 (37) niedrig (<15.1)

9. L512Q (61) (Nishihara et al., 2006) 4.5±0.7 niedrig (<15.1)

10. I568T (85) (Tonacchera et al., 2000) 25.6±6.3 hoch (≥15.1)

11. I568T (94) (Watkins et al., 2007) 25.6±6.3 hoch (≥15.1)

12. V597L (20) (Esapa et al., 1999) 125.2±10.5 (55) hoch (≥15.1)

13. F631L (45) (Kopp et al., 1995) 45.9±9.4 hoch (≥15.1)

14. T632I (43) (Kopp et al., 1997) 14.5±2.7 niedrig (<15.1)

15. D633Y (14) (Bircan et al, 2007) 16.4±6.4 hoch (≥15.1)

16. I630L (6) (Bertalan et al., 2010) keine LRA Daten -

46

VIII. Selbstständigkeitserklärung

Erklärung über die eigenständige Abfassung der Arbe it

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig und ohne unzulässige Hilfe

oder Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Ich versichere,

dass Dritte von mir weder unmittelbar noch mittelbar geldwerte Leistungen für Arbeiten

erhalten haben, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation ste-

hen, und dass die vorgelegte Arbeit weder im Inland noch im Ausland in gleicher oder

ähnlicher Form einer anderen Prüfungsbehörde zum Zweck einer Promotion oder eines

anderen Prüfungsverfahrens vorgelegt wurde. Alles aus anderen Quellen und von ande-

ren Personen übernommene Material, das in der Arbeit verwendet wurde oder auf das di-

rekt Bezug genommen wird, wurde als solches kenntlich gemacht. Insbesondere wurden

alle Personen genannt, die direkt an der Entstehung der vorliegenden Arbeit beteiligt wa-

ren.

................................. ....................................

Datum Unterschrift

47

IX. Lebenslauf

Persönliche Angaben

Name Julia Cordula Lüblinghoff

Geburtsdatum 8. Juli 1983

Geburtsort Iserlohn (Nordrhein-Westfalen)

Staatsangehörigkeit deutsch

Familienstand ledig

Bildungsweg

Juni 2002 Abitur am Gymnasium An der Stenner, Iserlohn

2002– 2009 Studium der Humanmedizin an der Universität Leipzig

August 2004 Ärztliche Vorprüfung

Oktober 2009 Zweiter Abschnitt der Ärztlichen Prüfung

Dezember 2009 Approbation als Ärztin

Dissertation

seit Oktober 2006 „Klinische Charakterisierung von TSH-Rezeptor Mutationen“ Universitätsklinikum Leipzig Klinik und Poliklinik für Endokrinologie und Nephrologie betreut von Prof. Dr. Ralf Paschke

Beruflicher Werdegang

seit Mai 2010 Beginn des Commun Trunk der Weiterbildung zur Fachärz-tin für Orthopädie und Unfallchirurgie in der Klinik für Endoprothetik an den Sana Kliniken Sommerfeld bei Herrn PD Dr. Halder

48

X. Publikationsliste

2007 Wissenschaftliches Poster und Vortrag: LÜBLINGHOFF J, MÜLLER S SONTHEIMER J AND PASCHKE R „Lack of consistent association of TSH Receptor Mutations in vitro Activity with the clinical cour-se of patients with sporadic non-autoimmune hyperthyroidism“ 32nd Ann. Meeting of the European Thyroid Association, Leipzig

2008 Wissenschaftliches Poster: LÜBLINGHOFF J, MÜLLER S, JÄSCHKE H, ESZLINGER M, BERGER H, BIRCAN R, GÖZU H, SANCAK S AND PASCHKE R „Different in vitro activities of sporadic, familiar and somatic TSH-Receptor mutations” 33rd Ann. Meeting of the Eu-ropean Thyroid Association, Thessaloniki, Griechenland

2009 Wissenschaftliche Poster: LÜBLINGHOFF J, MÜLLER S, JÄSCHKE H, ESZLINGER M, BIRCAN R, GÖZU H, SANCAK S AND PASCHKE R „Lack of genotype / phenotype correlations for somatic TSH-receptor mutations in hot thyroid nodules“ 34th Ann. Meeting of the European Thyroid Association, Lissabon, Portugal und: 53. Symposium der deutschen Gesellschaft für Endokrinologie Leipzig, Deutschland

MUELLER S, GOZU HI, BIRCAN R, JAESCHKE H, ESZLINGER M, LUEBLINGHOFF J, KROHN K AND PASCHKE R „Cases of borderline in vitro constitutive thyrotropin receptor activity: how to decide whether a thyrotropin receptor mutation is constitutively active or not?“ Thyroid. 2009 19:765-73.

GÖZU H, LÜBLINGHOFF J, BIRCAN R AND PASCHKE R „Non-autoimmune Auto-somal Dominant Hyperthyroidism“ Encyclopedia of Molecular Mechanisms of Disease. 2009 8: 945-948

2010 LÜBLINGHOFF J, MÜLLER S, SONTHEIMER J AND PASCHKE R „Lack of Con-sistent Association of TSH Receptor Mutations In Vitro Activity with the Clini-cal Course of Patients with Sporadic Non-autoimmune Hyperthyroidism“ Journal of Endocrinological Investigation. 2010 33: 228-33.

GOZU HI, LÜBLINGHOFF J, BIRCAN R AND PASCHKE R „Genetics and pheno-mics of inherited and sporadic non-autoimmune hyperthyroidism“ Molecular and Cellular Endocrinology. 2010 322:125-134.

2011 LUEBLINGHOFF J, ESZLINGER M, JAESCHKE H, MUELLER S, BIRCAN R, GOZU H, SANCAK S, AKALIN S AND PASCHKE R „Shared sporadic and somatic thyrotro-pin receptor mutations display more active in vitro activities than familial thy-rotropin receptor mutations.” Thyroid. 2011 21:221-9

JAESCHKE H, ESZLINGER M, LUEBLINGHOFF J, COSLOVSKY R AND PASCHKE R „Prolonged inappropriate TSH suppression during hypothyroidism after thy-roid ablation in a patient with nonautoimmune familial hyperthyroidism.” Hormone and Metabolic Research. 2011 43:500-4

49

XI. Danksagung

Hiermit möchte ich mich bei allen bedanken, die mir bei der Entstehung und der Fertig-

stellung dieser Arbeit geholfen haben.

Ein großes Dankeschön an Prof. Dr. R. Paschke für die Überlassung des Themas und die

exzellente Betreuung, außerdem an Dr. Sandra Huth und Dr. Holger Jäschke für die Hilfe

bei der in vitro Charakterisierung und die Beantwortung zahlreicher Fragen rund um den

TSH-Rezeptor.

Vielen Dank an Dr. Hilmar Berger und Dr. Markus Eszlinger.

Meinen Eltern danke ich vielmals für den Ansporn und die tolle Unterstützung, die zum

Abschluss dieser Arbeit geführt haben.

Als letztes möchte ich meiner Schwester und meinen Freunden dafür danken, dass sie

nicht nur sporadisch sondern konstitutiv aktiv bei allen autonomen und somatischen Fra-

gestellungen eine große Stütze sind.

klinische charakterisierung von tsh-rezeptormutationen · tsh thyreoidea stimulierendes hormon . 3...

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