Germline Mutation in TITF-1/NKX2.1 in Multinodular Goiter Thyroid Carcinoma
Germline Mutation in TITF-1/NKX2.1 in Multinodular Goiter Thyroid Carcinoma
Background: The genetic factors that determine the risk of papillary thyroid carcinoma (PTC) among patients with multinodular goiter (MNG) remain undefined. Because thyroid transcription factor-1 (TTF-1) is important to thyroid development, we evaluated whether the gene that encodes it, TITF-1/NKX2.1, is a genetic determinant of MNG/PTC predisposition.
Methods: Twenty unrelated PTC patients with a history of MNG (MNG/PTC), 284 PTC patients without a history of MNG (PTC), and 349 healthy control subjects were screened for germline mutation(s) in TITF-1/NKX2.1 by sequencing of amplified DNA from blood. The effects of the mutation on the growth and differentiation of thyroid cells were demonstrated by ectopic expression of wild-type (WT) and mutant proteins in PCCL3 normal rat thyroid cells, followed by tests of cell proliferation, activation of cell growth pathways, and transcription of TTF-1 target genes. All statistical tests were two-sided.
Results: A missense mutation (1016C> T) was identified in TITF-1/NKX2.1 that led to a mutant TTF-1 protein (A339V) in four of the 20 MNG/PTC patients (20%). These patients developed substantially more advanced tumors than MNG/PTC or PTC patients without the mutation (P =.022, Fisher exact test). Notably, this germline mutation was dominantly inherited in two families, with some members bearing the mutation affected with MNG, associated with either PTC or colon cancer. The mutation encoding the A339V substitution was not found among the 349 healthy control subjects nor among the 284 PTC patients who had no history of MNG. Overexpression of A339V TTF-1 in PCCL3 cells, as compared with overexpression of WT TTF-1, was associated with increased cell proliferation including thyrotropin-independent growth (average A339V proliferation rate = 134.27%, WT rate = 104.43%, difference = 34.3%, 95% confidence interval = 12.0% to 47.7%, P =.010), enhanced STAT3 activation, and impaired transcription of the thyroid-specific genes Tg, TSH-R, and Pax-8.
Conclusion: This is the first germline mutation identified in MNG/PTC patients. It could contribute to predisposition for MNG and/or PTC and to the pathogenesis of PTC.
Thyroid cancer is among the 10 most common malignancies and occurs more often in women than in men. Approximately 80% of all primary thyroid cancers consist of papillary thyroid carcinoma (PTC). Somatic genetic alterations in RET/PTC, BRAF, or RAS account for approximately 70% of all PTC cases and may contribute to a cancer initiation process that is influenced by the germline DNA inheritance of the patient. Emerging evidence indicates a possible progression from benign thyroid lesions to malignant disease, especially from multinodular goiter (MNG) to PTC. This notion has been supported by the existence of pedigrees whose members are affected with MNG, PTC, or both. In addition, in familial papillary thyroid carcinoma (FPTC), autosomal dominant inheritance of a predisposition to MNG, PTC, or both further suggests the presence of common genetic determinants for these two diseases. Nevertheless, the genetic etiology of MNG/PTC is yet unknown.
The search for genetic determinants underlying the hereditability and pathogenicity of FPTC has thus far revealed four putative susceptibility loci (TCO at 19p13.2, MIM 603386 [OMIM]; MNG1 at 14q, MIM 138800 [OMIM]; PRN1 at 1p13.2-1q22, MIM 605642 [OMIM]; and NMTC1 at 2q21, MIM 606240 [OMIM]), although the causative genes have not yet been identified. Additional studies conducted on families segregating FPTC failed to detect linkage to the above loci and thereby suggested that additional genes are involved. In addition, the identification of somatic mutations in FPTC suggests that inherited genetic determinants can predispose to somatic mutations in the tissue that would contribute to tumorigenesis.
TITF-1/NKX2.1 maps to chromosome 14q13. This gene comprises two exons that encode the 42-kDa thyroid transcription factor-1 (TTF-1) protein. The TTF-1 protein is a 371 amino acid transcription factor that includes two independent transcription activation domains located on either side of a central DNA-binding homeodomain. It activates transcription of the thyroglobulin, thyroperoxidase, and thyrotropin receptor genes, and thus is implicated in thyroid function. Genetic deletion of Titf-1/Nkx2.1 in mice results in embryonic lethality and several congenital defects that include absence of a thyroid gland. In addition, in a line of mice in which Titf-1/Nkx2.1 was conditionally deleted by expression of Cre recombinase under the human thyroid peroxidase (TPO) gene promoter (T/ebp/Nkx2.1(fl/fl);TPO-Cre), Titf-1/Nkx2.1 became inactivated in 85% of adult (but not embryonic) thyroid follicular cells. Interestingly, these mice exhibited either atrophic/degenerative thyroid follicles with frequent presence of adenomas and extremely high serum TSH levels or an altered thyroid structure with reduced numbers of extremely dilated follicles each composed of more than the usual numbers of follicular cells. These findings further suggest that TTF-1 is required for the maintenance of the normal architecture and function of the differentiated thyroid. In humans, TITF-1/NKX2.1 mutations are associated with respiratory distress, benign hereditary chorea, and congenital hypothyroidism. Importantly, TITF-1/NKX2.1 is also known to be expressed at lower levels in malignant thyroid as compared with normal thyroid tissue and is used as a marker of thyroid differentiation.
Given the relevance of TTF-1 to thyroid development and its potential role in thyroid cancer, we hypothesized that TITF-1/NKX2.1 could be a susceptibility gene that confers predisposition to MNG and/or PTC. In this study, we aimed to 1) identify potential TITF-1/NKX2.1 variant(s) implicated in MNG/PTC, 2) evaluate the incidence of such genetic variant(s) among MNG/PTC and PTC patients, and 3) elucidate the biological relevance of the variant(s) in the pathogenesis of MNG/PTC. To do this, we genetically screened 20 patients with MNG and PTC, 284 PTC patients, and 349 healthy individuals. We also assessed the functional consequences of this mutation in the growth and differentiation of the thyroid cells by overexpression of the mutant and wild-type (WT) TTF-1 proteins in normal rat thyroid cells and comparison of cell proliferation, activation of growth pathways, and transcription of TTF-1 target genes.
Abstract and Introduction
Abstract
Background: The genetic factors that determine the risk of papillary thyroid carcinoma (PTC) among patients with multinodular goiter (MNG) remain undefined. Because thyroid transcription factor-1 (TTF-1) is important to thyroid development, we evaluated whether the gene that encodes it, TITF-1/NKX2.1, is a genetic determinant of MNG/PTC predisposition.
Methods: Twenty unrelated PTC patients with a history of MNG (MNG/PTC), 284 PTC patients without a history of MNG (PTC), and 349 healthy control subjects were screened for germline mutation(s) in TITF-1/NKX2.1 by sequencing of amplified DNA from blood. The effects of the mutation on the growth and differentiation of thyroid cells were demonstrated by ectopic expression of wild-type (WT) and mutant proteins in PCCL3 normal rat thyroid cells, followed by tests of cell proliferation, activation of cell growth pathways, and transcription of TTF-1 target genes. All statistical tests were two-sided.
Results: A missense mutation (1016C> T) was identified in TITF-1/NKX2.1 that led to a mutant TTF-1 protein (A339V) in four of the 20 MNG/PTC patients (20%). These patients developed substantially more advanced tumors than MNG/PTC or PTC patients without the mutation (P =.022, Fisher exact test). Notably, this germline mutation was dominantly inherited in two families, with some members bearing the mutation affected with MNG, associated with either PTC or colon cancer. The mutation encoding the A339V substitution was not found among the 349 healthy control subjects nor among the 284 PTC patients who had no history of MNG. Overexpression of A339V TTF-1 in PCCL3 cells, as compared with overexpression of WT TTF-1, was associated with increased cell proliferation including thyrotropin-independent growth (average A339V proliferation rate = 134.27%, WT rate = 104.43%, difference = 34.3%, 95% confidence interval = 12.0% to 47.7%, P =.010), enhanced STAT3 activation, and impaired transcription of the thyroid-specific genes Tg, TSH-R, and Pax-8.
Conclusion: This is the first germline mutation identified in MNG/PTC patients. It could contribute to predisposition for MNG and/or PTC and to the pathogenesis of PTC.
Introduction
Thyroid cancer is among the 10 most common malignancies and occurs more often in women than in men. Approximately 80% of all primary thyroid cancers consist of papillary thyroid carcinoma (PTC). Somatic genetic alterations in RET/PTC, BRAF, or RAS account for approximately 70% of all PTC cases and may contribute to a cancer initiation process that is influenced by the germline DNA inheritance of the patient. Emerging evidence indicates a possible progression from benign thyroid lesions to malignant disease, especially from multinodular goiter (MNG) to PTC. This notion has been supported by the existence of pedigrees whose members are affected with MNG, PTC, or both. In addition, in familial papillary thyroid carcinoma (FPTC), autosomal dominant inheritance of a predisposition to MNG, PTC, or both further suggests the presence of common genetic determinants for these two diseases. Nevertheless, the genetic etiology of MNG/PTC is yet unknown.
The search for genetic determinants underlying the hereditability and pathogenicity of FPTC has thus far revealed four putative susceptibility loci (TCO at 19p13.2, MIM 603386 [OMIM]; MNG1 at 14q, MIM 138800 [OMIM]; PRN1 at 1p13.2-1q22, MIM 605642 [OMIM]; and NMTC1 at 2q21, MIM 606240 [OMIM]), although the causative genes have not yet been identified. Additional studies conducted on families segregating FPTC failed to detect linkage to the above loci and thereby suggested that additional genes are involved. In addition, the identification of somatic mutations in FPTC suggests that inherited genetic determinants can predispose to somatic mutations in the tissue that would contribute to tumorigenesis.
TITF-1/NKX2.1 maps to chromosome 14q13. This gene comprises two exons that encode the 42-kDa thyroid transcription factor-1 (TTF-1) protein. The TTF-1 protein is a 371 amino acid transcription factor that includes two independent transcription activation domains located on either side of a central DNA-binding homeodomain. It activates transcription of the thyroglobulin, thyroperoxidase, and thyrotropin receptor genes, and thus is implicated in thyroid function. Genetic deletion of Titf-1/Nkx2.1 in mice results in embryonic lethality and several congenital defects that include absence of a thyroid gland. In addition, in a line of mice in which Titf-1/Nkx2.1 was conditionally deleted by expression of Cre recombinase under the human thyroid peroxidase (TPO) gene promoter (T/ebp/Nkx2.1(fl/fl);TPO-Cre), Titf-1/Nkx2.1 became inactivated in 85% of adult (but not embryonic) thyroid follicular cells. Interestingly, these mice exhibited either atrophic/degenerative thyroid follicles with frequent presence of adenomas and extremely high serum TSH levels or an altered thyroid structure with reduced numbers of extremely dilated follicles each composed of more than the usual numbers of follicular cells. These findings further suggest that TTF-1 is required for the maintenance of the normal architecture and function of the differentiated thyroid. In humans, TITF-1/NKX2.1 mutations are associated with respiratory distress, benign hereditary chorea, and congenital hypothyroidism. Importantly, TITF-1/NKX2.1 is also known to be expressed at lower levels in malignant thyroid as compared with normal thyroid tissue and is used as a marker of thyroid differentiation.
Given the relevance of TTF-1 to thyroid development and its potential role in thyroid cancer, we hypothesized that TITF-1/NKX2.1 could be a susceptibility gene that confers predisposition to MNG and/or PTC. In this study, we aimed to 1) identify potential TITF-1/NKX2.1 variant(s) implicated in MNG/PTC, 2) evaluate the incidence of such genetic variant(s) among MNG/PTC and PTC patients, and 3) elucidate the biological relevance of the variant(s) in the pathogenesis of MNG/PTC. To do this, we genetically screened 20 patients with MNG and PTC, 284 PTC patients, and 349 healthy individuals. We also assessed the functional consequences of this mutation in the growth and differentiation of the thyroid cells by overexpression of the mutant and wild-type (WT) TTF-1 proteins in normal rat thyroid cells and comparison of cell proliferation, activation of growth pathways, and transcription of TTF-1 target genes.