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. PATIENTS: 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.

Genetic analysis of TTF-2 gene in children with congenital hypothyroidism and cleft palate, congenital hypothyroidism, or isolated cleft palate.

Homozygous null mice for thyroid transcription factor (TTF)-2 gene exhibit cleft palate and thyroid malformation. We performed a genetic analysis of the TTF-2 gene in 2 children with congenital hypothyroidism (CH) and cleft palate, 45 children with thyroid dysgenesis, 19 children with isolated cleft palate or cleft lip, 4 patients with thyroid hemiagenesis. The entire coding-region of the TTF-2 gene was analyzed by direct sequencing. Direct sequencing of the TTF-2 gene revealed polymorphisms in the length of the polyalanine tract. The most frequent stretch length was 14 residues and it was found in 50 of 70 (71%) and in 45 of 53 (85%) normal healthy controls. A polyalanine tract of 16 residues in the heterozygous state was seen in 18 of 70 (26%) cases and in 4 of 53 (7%) normal subjects. In 1 of 4 (25%) case of hemiagenesis a polyalanine tract of 16 residues in the homozygous state was observed. In 1 of 26 agenesis the polyalanine tract consisted of 12 residues in the heterozygous state. Direct sequencing also revealed the presence of two silent polymorphisms. No mutations were identified in the TTF-2 gene. In conclusion, our results show that no genetic alteration was present in the TTF-2 gene of these patients, suggesting that defects in the TTF-2 gene are a rare event.