An infant girl was born at 37 weeks gestation and found to be clinically thyrotoxic at 9 months of age. Thyroid autoantibodies were negative, and thyroid function failed to normalize with medical treatment. The patient underwent a total thyroidectomy. DNA obtained from her thyroid gland and leukocytes was analyzed for thyrotropin receptor (TSHR) mutations using single strand conformation polymorphism and direct sequencing. A mobility shift of polymerase chain reaction (PCR)-amplified DNA was detected on single strand conformation polymorphism gel. Direct sequencing identified a novel point mutation in the fifth transmembrane domain of the TSH receptor at codon 597 (GTC to CTC), resulting in the amino acid substitution of leucine for valine. The mutation was heterozygous and germline, and was not identified in DNA from either of her parents. Expression of the V597L mutant is transiently transfected COS 7 cells displayed increased constitutive cyclic adenosine monophosphate (cAMP) production compared with the wild-type receptor. The mutant is expressed at very low levels on the surface of COS cells, and its response to TSH is marginal.

Objective: The adenylyl cyclase system plays an important role in the control of both thyroid follicular and anterior pituitary cell function. Activating mutations affecting important pathway components such as the TSH receptor and Gsa occur in the majority of autonomously functioning thyroid nodules. Only a small proportion of other types of thyroid tumours, however, have been reported to harbour these mutations. Activating mutations of Gsa have been reported to occur in up to 40% of pituitary somatotroph adenomas. As the majority of cold thyroid nodules and pituitary tumours are unaffected by these mutations, we have investigated the possibility of activating mutations occurring in protein kinase A (PKA), which is another key component of the adenylyl cyclase pathway. Design: Genomic DNA and cDNA were analysed for the presence of PKA Ca mutations by allele-specific oligonucleotide hybridisation and single strand conformation polymorphism analysis. Patients: A total of 171 tissue samples were investigated. These comprised 66 benign and 24 malignant thyroid neoplasms, 21 somatotroph adenomas, 35 non-functioning pituitary adenomas, 2 corticotroph adenomas, 1 malignant prolactinoma, and 22 normal pituitary tissue samples. Results: No mutations of PKA Ca were identified using either allele-specific oligonucleotide hybridisation or single strand conformation polymorphism analysis.Conclusions: It appears that PKA Ca mutations at the codons investigated do not represent an oncogenetic mechanism in the development of thyroid and pituitary neoplasms.

Homozygosity for the C282Y mutation of the HFE gene is a highly significant risk factor for the development of hereditary hemochromatosis (HH) and the majority of patients with HH have this genotype. An Irish/Belgian female with an elevated serum ferritin level and a family history of hemochromatosis was tested for the presence of the C282Y and H63D mutations. Results of digested PCR products have shown the patient to be homozygous for C282Y mutation and heterozygous for H63D mutation. Sequencing confirmed these findings. Genotyping of the patient's offspring and husband has also indicated the inheritance of both C282Y and H63D in 'cis'. Implications of this finding are: 1) the compound heterozygous state is by far the most common, but not the universal, phase for individuals found to be heterozygous for the two mutations, C282Y and H63D; 2) the C282Y and H63D mutations in the 'cis' phase may account for some cases of questionable parentage.

The cAMP pathway plays a central role in thyroid follicular cell growth and function. Mutations of the TSH receptor (TSHR) or G proteins (gsp) that activate adenylyl cyclase have been identified in autonomously functioning thyroid nodules. Gsp mutations have been identified also in other forms of thyroid neoplasia, but their reported prevalence has been extremely variable. We have studied the prevalence of gsp mutations and activating mutations of Gi2 alpha (gip) in a series of 66 benign and 34 malignant thyroid tumors. Thirty-six tumors were from Boston and 64 from the UK. In addition, we examined the 64 UK tumors for mutations of the TSHR gene. DNA extracted from fresh-frozen or paraffin-embedded tissue was amplified by PCR and examined for mutations using oligonucleotide-specific hybridization and single-strand conformation polymorphism analysis. No G protein gene mutations were identified in the Boston tumors. One gsp mutation, R201C, in a Hürthle cell adenoma and 1 gip mutation, R179C, in a follicular adenoma were demonstrated in tumors from the UK. Oligonucleotide-specific hybridization and single-strand conformation polymorphism analysis of the UK tumors did not demonstrate any mutations of the TSHR gene. Eleven normal thyroid tissue samples were wild-type for Gs alpha, Gi2 alpha, and the TSHR gene.