The p.A2215D thyroglobulin gene mutation leads to deficient synthesis and secretion of the mutated protein and congenital hypothyroidism with wide phenotype variation.

CONTEXT: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. OBJECTIVES: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p.A2215D mutation. DESIGN: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. RESULTS: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p.A2215D, p.R277X, and g.IVS30+1G>T), and two novel mutations (p.Q2142X and g.IVS46-1G>A). Two known (g.IVS30+1G/p.A2215D and p.A2215D/p.R277X) and one novel (p.R277X/g.IVS46-1G>A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. CONCLUSION: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p.A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone.

A molecular analysis and long-term follow-up of two siblings with severe congenital hypothyroidism carrying the IVS30+1G>T intronic thyroglobulin mutation.

OBJECTIVE: To extend the molecular analysis of the IVS30+1G>T intronic thyroglobulin (TG) mutation, and to report the eleven year follow-up of the affected patients. METHOSD: Two siblings with severe congenital hypothyroidism with fetal and neonatal goiter, harboring the IVS30+1G>T mutation were included. Nodular and non-nodular thyroid tissue specimens were collected. Specific thyroid genes expression was evaluated by real-timePCR and by immunohistochemistry. RESULTS: In non-nodular tissue specific thyroid genes mRNA were reduced when compared to normal thyroid sample. In the nodule, TPO and NIS expression was very low. Microscopic examinations showed very large follicular-lumina and swollen vesicles of endoplasmatic-reticulum. Strong cytoplasmatic and low follicular-lumen TG immunostaining were detected. Intracellular NIS, membrane TPO and TSHR immunostaining had higher positivity in non-nodular sample. Both patients had a long-term adequate developmental outcome, besides one patient have been lately-treated. CONCLUSIONS: IVS30+1G>T mutation not only lead to very enlarge endoplasmatic-reticulum, but also to alterations of specific thyroid genes expression. The clinical evolution of patients harboring these mutations strengthen the concept of the influence of environment, like iodine nutrition, to determine the final phenotypic appearance.

A molecular analysis and long-term follow-up of two siblings with severe congenital hypothyroidism carrying the IVS30+1G>T intronic thyroglobulin mutation / Análise molecular e acompanhamento a longo prazo de dois irmãos com hipotireoidismo congênito portadores da mutação intrônica IVS30+1G>T no gene da tireoglobulina

OBJECTIVE: To extend the molecular analysis of the IVS30+1G>T intronic thyroglobulin (TG) mutation, and to report the eleven year follow-up of the affected patients. METHOSD: Two siblings with severe congenital hypothyroidism with fetal and neonatal goiter, harboring the IVS30+1G>T mutation were included. Nodular and non-nodular thyroid tissue specimens were collected. Specific thyroid genes expression was evaluated by real-timePCR and by immunohistochemistry. RESULTS: In non-nodular tissue specific thyroid genes mRNA were reduced when compared to normal thyroid sample. In the nodule, TPO and NIS expression was very low. Microscopic examinations showed very large follicular-lumina and swollen vesicles of endoplasmatic-reticulum. Strong cytoplasmatic and low follicular-lumen TG immunostaining were detected. Intracellular NIS, membrane TPO and TSHR immunostaining had higher positivity in non-nodular sample. Both patients had a long-term adequate developmental outcome, besides one patient have been lately-treated. CONCLUSIONS: IVS30+1G>T mutation not only lead to very enlarge endoplasmatic-reticulum, but also to alterations of specific thyroid genes expression. The clinical evolution of patients harboring these mutations strengthen the concept of the influence of environment, like iodine nutrition, to determine the final phenotypic appearance.

OBJETIVO: Aprofundar a análise molecular da mutação intrônica IVS30+1G>T do gene tireoglobulina (TG) e relatar a clínica de pacientes portadores da mutação, acompanhados por 11 anos. MÉTODOS: Foram estudados dois irmãos com hipotireoidismo congênito grave com bócio fetal e bócio neonatal, portadores da mutação IVS30+1G>T. Foram coletadas amostras de tecido nodular e não-nodular. Avaliou-se a expressão de genes específicos da tireóide por PCR em tempo real e imunohistoquímica. RESULTADOS: A expressão de genes específicos da tireóide foi menor no tecido não-nodular que no tecido normal controle. Expressões de TPO e NIS foram extremamente baixas no tecido nodular. Verificou-se lúmen folicular aumentado com grandes vesículas de retículo endoplasmático, e detectou-se forte marcação de TG no citoplasma e fraca no lúmen folicular. No tecido não-nodular observou-se forte positividade de NIS intracelular e, TPO e TSHR na membrana plasmática. O acompanhamento em longo prazo dos pacientes mostrou adequado desenvolvimento, apesar de um deles ter recebido tratamento tardio. CONCLUSÕES: A mutação IVS30+1G>T não só promove alterações no retículo endoplasmático, como alterações na expressão de genes específicos da tireóide. A evolução clínica destes pacientes reforça o conceito da influência do meio ambiente, como o aporte nutricional de iodo, no fenótipo final.