Cases of borderline in vitro constitutive thyrotropin receptor activity: how to decide whether a thyrotropin receptor mutation is constitutively active or not?
BACKGROUND
Previous in vitro data for several constitutively activating thyrotropin receptor (TSHR) mutations reported divergent results for the constitutive activity of the same mutations. Moreover, several case reports have highlighted the difficulties in determining whether a TSHR mutation is constitutively active or not. Retrospectively, this has repeatedly been the case for mutants with only a slight increase of basal cAMP activity. We re-examined 10 previously described TSHR germline mutations with minor increases of basal cAMP activity and analyzed the influences of the cell line and vector system on the basal receptor activity.


METHODS
TSHR mutations were characterized by determination of cell surface expression, cAMP accumulation, and linear regression analysis of constitutive activity.


RESULTS
Re-examination of the previously described constitutively active TSHR germline mutations did not show constitutive activity for R310C and N670S as tested in COS-7 cells and confirmed constitutive activity for the other eight mutations. However, mutant N670S showed a slight but significant increase of basal activity measured by linear regression analysis when analyzed in HEK(GT) cells transiently transfected with pcDNA but not with the pSVL vector. This was not the case for R310C.


CONCLUSIONS
Our findings indicate that current methods to precisely classify mutants with only a slight increase of the basal activity as constitutively active are limited. The results concerning the level of the basal activity can be influenced by the vector and/or the cell system. A comprehensive clinical characterization of the respective patients appears as a necessary and promising adjunct for the activity classification of these borderline mutations.
Mueller
Sandra
S
Department of Internal Medicine III, University of Leipzig, Leipzig, Germany.
Gozu
Hulya Iliksu
HI
Bircan
Rifat
R
Jaeschke
Holger
H
Eszlinger
Markus
M
Lueblinghoff
Julia
J
Krohn
Knut
K
Paschke
Ralf
R
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D004252:DNA Mutational Analysis; D018095:Germ-Line Mutation; D006801:Humans; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000378:metabolism; D012044:Regression Analysis; D014162:Transfection
D011989:Receptors, Thyrotropin; D000242:Cyclic AMP