Screening and functional analysis of thyroglobulin gene mutations de mutações related to congenital goiter and hypothyroidism / Rastreamento e estudo funcional de mutações no gene da tireoglobulina associadas a bócio congênito e hipotireoidismo

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Introduction: Congenital hypothyroidism is one of the most common hereditary endocrine disorders, which affects 1:4000 newborns. Congenital hypothyroidism is caused by thyroid gland dysgenesis (80%) or inborn errors of thyroid hormone synthesis (20%). Genetic defects in thyroglobulin, pendrin, thyroperoxidase, dual oxidase 2, simporter sodium/iodine have been associated to dyshormonogenesis. Thyroglobulin is a large glycoprotein that functions as the matrix for thyroid hormone synthesis. At least 38 mutations in the TG gene have been identified in patients with thyroid dyshormonogenesis. Objectives: The aims of this study were to identify thyroglobulin gene mutations associated with congenital hypothyroidism in 13 Brazilian patients, and to determine the functional effect of the mutation A2215D identified in this study. Patients and methods: Thirteen patients with congenital hypothyroidism due to defective thyroglobulin synthesis were included. Peripheral blood DNA from all the patients and one thyroid tissue sample from a patient with the A2215D mutation were collected. Thyroglobulin exons and exons/introns borders were amplified by PCR and sequenced. Mammalian cells were transfected with expression vectors encoding mutated and non-mutated thyroglobulin cDNA. Immunoblots, determination of thyroglobulin concentrations, real time PCR to quantify mRNA expression, immunohistochemical analysis, hematoxilyn-eosin staining and electronic and immunogold microscopy were performed. Results Abnormal thyroglobulin synthesis and secretion was confirmed by the absence of a serum thyroglobulin elevation 24 and 48 hours after the stimulation with recombinant human TSH (0.45 mg). Molecular analysis revealed five mutations in the thyroglobulin gene, 2 novel (Q2142X and IVS46-1G>A) and three previously described mutations (R277X, IVS30+1G>T and A2215D); 19 polymorphisms and 13 intronic alterations. Biallelic mutations (homozygous or compound heterozygous) in the thyroglobulin gene were identified in twelve of thirteen patients. Functional studies in mammallian cells showed low secretion of the mutated thyroglobulin (A2215D). The complete analysis of the thyroid tissue from a patient with the A2215D mutation revealed: mutant thyroglobulin in the follicular cell but not in the lumen, marked dilatation of the endoplasmic reticulum, thyroglobulin immunopositivity in the endoplasmic reticulum and low concentration of thyroglobulin protein and mRNA. Furthermore, low mRNA levels of thyroid genes (TPO, TTF1, PAX-8, NIS, receptor de TSH) were detected. Conclusions: All the identified thyroglobulin gene mutations explained the congenital goiter hypothyroidism of the patients. The mutation A2215D promoted the retention of the mutant thyroglobulin in the endoplasmic reticulum, decreased the thyroglobulin secretion to the colloid resulting in an impairment of thyroid hormone synthesis and congenital hypothyroidism. The endogenous A2215D mutant thyroglobulin could be considered a regulator of follicular function mediated by the transcriptional suppression of thyroid genes.

ASSUNTO(S)

thyroglobulin mutação mutations congenital hypothyroidism hipotireoidismo congênito tireoglobulina

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