Identification of TSH receptor mutations in three families with resistance to TSH.
OBJECTIVE
Genetic analysis of the TSH receptor gene in seven subjects with subclinical hypothyroidism (SH), in whom the diagnosis of autoimmune thyroid disease had been excluded by laboratory and instrumental techniques currently available.


METHODS
Three families where different members (2 children and 5 adults) affected by SH were studied. GENETIC ANALYSIS: Genomic DNA was extracted from peripheral lymphocytes and the entire coding sequence of the TSHr gene was sequenced. pSVL-TSHr construct harbouring a Q8fsX62 insertion was obtained by site-directed mutagenesis. COS-7 cells transfected with wild-type and mutant receptor were used for binding studies, flow cytometry, and cyclic AMP (cAMP) determination.


RESULTS
A four base pair (bp) duplication in position 41 (41TGCAins), leading to a premature stop of translation at codon 62 (Q8fsX62), was found to be heterozygous in the proband, the father and the sister in Family 1. In Family 2 the proband and the sister were heterozygous for the mutation D410N. In Family 3 the proband and the father were heterozygous for the mutation P162A. After transfection in COS-7 cells, the mutant receptor Q8fsX62 displayed a low expression at the cell surface, and a reduced response to bovine TSH (bTSH) in terms of cAMP production.


CONCLUSIONS
We identified TSH receptor mutations in seven members of three families with subclinical hypothyroidism.
Tonacchera
Massimo
M
Dipartimento di Endocrinologia e Metabolismo, Centro Eccellenza AmbiSEN, Università di Pisa, Pisa, Italy. mtonacchera@hotmail.com
Di Cosmo
Caterina
C
De Marco
Giuseppina
G
Agretti
Patrizia
P
Banco
Mariaelena
M
Perri
Anna
A
Gianetti
Elena
E
Montanelli
Lucia
L
Vitti
Paolo
P
Pinchera
Aldo
A
D000293:Adolescent; D000328:Adult; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002648:Child; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010375:Pedigree; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000378:metabolism; D018382:Thyroid Hormone Resistance Syndrome / Q000097:blood / Q000235:genetics*; D013972:Thyrotropin / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone; D013974:Thyroxine / Q000097:blood; D014162:Transfection / Q000379:methods; D014284:Triiodothyronine / Q000097:blood
D011989:Receptors, Thyrotropin; D014284:Triiodothyronine; D013973:Thyrotropin-Releasing Hormone; D013972:Thyrotropin; D000242:Cyclic AMP; D013974:Thyroxine