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 / 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.

Phenotypic variation among four family members with congenital hypothyroidism caused by two distinct thyroglobulin gene mutations.

BACKGROUND: Thyroglobulin (Tg) is a large glycoprotein that is intimately involved in the biosynthesis of thyroxine and triiodothyronine. At least 38 mutations have been described in the Tg gene that are associated with varying degrees of hypothyroidism. We studied the Tg gene in four related subjects with congenital hypothyroidism. SUMMARY: We found a novel compound heterozygous constellation (IVS30 + 1G>T/A2215D) in a brother and sister and one previously described related mutation (IVS30+1G>T) in their two sibling second degree cousins. The brother with the IVS30 + 1G>T/A2215D mutation and the two siblings with the IVS30+1G>T mutation had fetal or neonatal goiter and all had hypothyroidism. CONCLUSIONS: This study further confirms the association of the IVS30+G>T mutation of the Tg gene with hypothyroidism. Computer analysis predicts that the A2215D mutation, first reported here, should cause structural instability of Tg but when present as a compound heterozygous mutation with IVS30+G>T/A its effect is unclear but is likely to be influenced by iodine intake.

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.

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

Introdução: O hipotireoidismo congênito possui prevalência de 1/4000 crianças nascidas vivas e pode ser causado por disgenesia tireoideana (80% dos casos) ou por defeitos de síntese hormonal (20% restantes). A disormonogênese tem sido associada a mutações nos genes: tireoglobulina, simportador sódio/iodo, tireoperoxidase, dual oxidase 2, e pendrina. A tireoglobulina é uma glicoproteína de 660KDa e funciona como matriz para a síntese dos hormônios tireoideanos. Até o momento 38 mutações inativantes, associadas a bócio e hipotireoidismo, foram identificadas no gene da tireoglobulina. Objetivos: Este estudo visou caracterizar mutações no gene da tireoglobulina em 13 pacientes brasileiros com bócio e hipotireoidismo congênito e verificar o efeito funcional da mutação A2215D identificada neste estudo. Casuística e Métodos: Foram estudados 13 pacientes com hipotireoidismo congênito por possível defeito de síntese de tireoglobulina. Foi utilizado DNA de sangue periférico de todos os pacientes e amostra de tecido da paciente portadora da mutação A2215D. Os métodos utilizados foram: amplificação e sequenciamento dos 48 exons e das junções exon/intron, transfecção de células de mamífero com plasmídeos contendo o cDNA da tireoglobulina mutada e não mutada, eletroforese de proteínas e hibridização com anticorpo específico, dosagem de tireoglobulina por fluoroimunoensaio indireto, quantificação de RNAm por PCR quantitativo em tempo real, imunohistoquímica, microscopia por coloração de hematoxilina/eosina e microscopia eletrônica e imunoeletrónica. Resultados: O defeito de síntese de tireoglobulina nos pacientes foi confirmado pela ausência de elevação do valor da tireoglobulina sérica 24 e 48hs após a aplicação de 0,45mg de TSH humano recombinante. No gene da tireoglobulina foram identificadas cinco mutações, sendo duas novas (Q2142X e IVS46-1G>A) e três já descritas na literatura (R277X, IVS30+1G>T e A2215D); 19 polimorfismos e 13 alterações em introns...

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)...