RESUMO
RESUMEN Objetivos Analizar las características clínicas, bioquímicas, estudios complementarios, hallazgos moleculares y la prevalencia de glándula eutópica en neonatos con HC pertenecientes al Programa Provincial de Pesquisa Neonatal de Córdoba, Argentina, entre 1996 y 2015. Analizar la evolución de los pacientes que reunieron criterios para una reevaluación. Pacientes y métodos Se analizaron retrospectivamente las historias clínicas de 237 pacientes detectados por pesquisa neonatal en la provincia de Córdoba, Argentina, entre 1996-2015 con una incidencia promedio de 1/2146 pesquisados. Presentaron glándula eutópica 81 pacientes (34%) F35/M46; se excluyeron 10 con síndromes genéticos asociados. Se analizaron los niveles de: TSH, T4T, T4L, T3, TPOAb / TGAb y Tiroglobulina (ECLIA -ROCHE) (VR: >15 días: 6-83 ng/ ml; <15 días: 29-173 ng/ml), ecografía y centellografía de cuello con Tc-99m. El valor de corte de TSH sérica adoptado para la confirmación diagnóstica fue de ≥10 mUI/ml. Se realizaron estudios de biología molecular en casos seleccionados. Se reevaluaron niños mayores de 3 años, sin bocio, con valores normales de Tiroglobulina y sin requerimiento de incrementos en la dosis de LT4. Resultados: La prevalencia de HC y Tiroides Eutópica se mantuvo constante. El 50% de los pacientes (36/71) mostraron hiperplasia glandular tiroidea. El 84% (n: 60 de 71) presentó niveles de TSH sérica ≥20 uUI/ml (20-1186) y el 75% (n: 53 de 71) >40 uUI/ml (40-1186). TGAb and TPOAb fueron positivos en un niño. La determinación de TG fue normal en el 29% (21/71) de los casos, elevada en el 56% (39/71) y baja en el 14% (10/71). Los estudios de biología molecular resultaron diagnósticos en 26 pacientes de 18 familias, demostrándose mutaciones en los genes de: TPO: 9 pacientes, TG: 12 pacientes, NIS: 2 pacientes, DUOX2: 2 pacientes y TRβ: 1 paciente. Se encontraron 11 nuevas mutaciones: tres en TPO, cinco en TG, dos en NIS y una en DUOX2. Se informaron anomalías congénitas en el 11% (8/71) de los pacientes. Se reevaluó el 11% (8/71) de los niños, resultando: HC transitorio n: 5, permanente n: 2 y una niña con Síndrome de Resistencia a las Hormonas Tiroideas. La prevalencia de lactantes con HC y glándula eutópica se mantuvo constante a lo largo de 19 años del Programa. Conclusiones Nuestros estudios demuestran que la prevalencia de Hipotiroidismo Congénito con glándula eutópica se mantuvo estable en los períodos analizados. Este grupo de pacientes se caracterizó predominantemente por presentar HC de carácter permanente acompañado por fenotipos de moderada a severa intensidad. En el futuro deberá profundizarse el conocimiento respecto a la influencia de factores medioambientales, como posibles agentes de riesgo asociados a la génesis de Hipotiroidismo Congénito.
abstract Objectives To describe clinical, biochemical characteristics and complementary studies to diagnosis, molecular findings and the prevalence of eutopic gland in newborn with CH detected through our neonatal screening program in Córdoba, Argentina, between 1996 and 2015. To analyze the evolution of the patients who met criteria for re-evaluation. Patients and methods We retrospectively analysed medical records of 237 patients with CH detected by neonatal screening in Córdoba, Argentina, from 1996 to 2015 with an average incidence of 1/2146 researched. 81 patients (34%) F35/M46 had eutopic thyroid gland; 10 patients with associated genetic syndromes were excluded. TT4, FT4, T3, TSH, TPOAb, TGAb and Thyroglobulin (VR: >15 days: 6-83 ng/ml; <15 days: 29-173 ng/ml) (ECLIA ROCHE), thyroid ultrasonography and 99Tc scan were assessed. The serum TSH cutoff value adopted for diagnostic confirmation was ≥10 mIU/ml. Molecular biology studies were performed in selected cases. Those who had no goiter, with normal thyroglobulin, and had not required increases in L-T4 dose underwent re-evaluation after the age of 3 years. Results The prevalence of HC and thyroid Eutopic remained constant. 50% of the patients (36/71) showed glandular hyperplasia. In 84% (60/71) presented serum TSH levels ≥20 uUI/ml (20-1186) and in 75% (n: 53 of 71) levels >40 uUI/ml (40-1186). TGAb and TPOAb were positive only in one baby. TG levels were: normal in 29% (21/71) of the cases, elevated in 56% (39/71) and low in 14% (10/71). Gene mutations were found in 26 patients from 18 families: TPO: 9 patients, TG: 12 patients, NIS: 2 patients, DUOX2: 2 patients y TRβ: 1 patient. Eleven new mutations were found: three in TPO, five in TG, two in NIS and one in DUOX2. Congenital anomalies were reported in 11% (8/71) patients. The 11% (8/71) of children were re-evaluated resulting in: 5 Transient CH, 2 Permanent CH and 1 with Resistance to Thyroid Hormones. The prevalence of infants with CH and eutopic gland remained constant along 19 years of the Program. Conclusions Our studies show that the prevalence of congenital hypothyroidism with eutopic gland remained stable in the periods analyzed. This group of patients was predominantly characterized by permanent CH accompanied by moderate to severe phenotypes. In the future, knowledge about the influence of environmental factors, as possible risk agents associated with the genesis of Congenital Hypothyroidism, should be deepened.
Assuntos
Humanos , Masculino , Feminino , Recém-Nascido , Glândula Tireoide/fisiopatologia , Hipotireoidismo Congênito/etiologia , Hipotireoidismo Congênito/fisiopatologia , Hormônios Tireóideos/genética , Anormalidades Congênitas/diagnóstico , Triagem Neonatal/métodos , Hiperplasia/genéticaRESUMO
Partial thyroxine-binding globulin deficiency (TBG-PD) is an endocrine defect with a prevalence of 1:4 000 in newborns. Due to the presence of a single TBG gene on the X chromosome, most familial TBG defects follow an X-linked inheritance pattern. Abnormal T4 binding to T4-binding prealbumin (TTR) is a rare cause of euthyroid hyperthyroxinemia, which is transmitted by autosomal dominant inheritance. The purpose of the present study was to identify and characterize new mutations in the Serpina7 and TTR genes in a complete family with typical TBG-PD. All patients underwent clinical and biochemical evaluation. Sequencing of DNA, population screening by (SSCP) analysis, and bioinformatics studies were performed. Molecular studies revealed a novel p.A64D mutation in the exon 1 of Serpina7 gene associated with the previously reported p.A109T mutation in the exon 4 of TTR gene. To our knowledge, this is the first report of a patient with a TBG-PD by a mutation in Serpina7 that was coincident with a mutation in TTR gene that increased affinity of TTR for T4. This work contributes to elucidate the molecular basis of the defects of thyroid hormone transport in serum and the improvement of the diagnosis avoiding unnecessary therapy.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação/genética , Pré-Albumina/genética , Pré-Albumina/metabolismo , Globulina de Ligação a Tiroxina/deficiência , Tiroxina/metabolismo , Sequência de Aminoácidos , Cromossomos Humanos X/genética , DNA/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto/genética , Linhagem , Pré-Albumina/química , Alinhamento de Sequência , Análise de Sequência de DNA , Globulina de Ligação a Tiroxina/química , Globulina de Ligação a Tiroxina/genéticaRESUMO
CONTEXT: Thyroglobulin (TG) gene mutations cause congenital hypothyroidism (CH) with goiter. A founder effect has been proposed for some frequent mutations. Mutated proteins have a defect in intracellular transport causing intracellular retention with ultrastructural changes that resemble an endoplasmic reticulum storage disease. OBJECTIVE: To reveal new aspects of thyroglobulin pathophysiology through clinical, cellular, molecular, and genetic studies in a family presenting with CH due to TG mutations from Galicia, an iodine-deficient area of Spain. DESIGN: The included clinical evaluation of family members, DNA sequencing for TG gene mutation and haplotyping analysis, ultrastructural analysis of thyroid tissue specimens from affected subjects, analysis of effects of mutations found on TG gene transcription, and in vitro studies of cellular production and secretion of mutated proteins. SETTING: Locations included primary care and university hospitals. RESULTS: Family members with CH, mental retardation, and goiter were compound heterozygous for c.886C-->T (p.R277X) and g.IVS35+1delG. For c.886C-->T, a founder effect cannot be excluded, and its transcription was hardly detectable. g.IVS35+1delG caused an in-frame deletion in exon 35 and produced a protein that, although synthesized, could not be secreted. Ultrastructural analyses showed morphological changes consistent with an endoplasmic reticulum storage disease. CONCLUSION: The shorter thyroglobulin resulting from the novel g.IVS35+1delG was retained within the endoplasmic reticulum of thyrocytes, and together with p.R227X caused severe hypothyroidism with goiter. p.R277X, the most commonly described TG mutation, is caused by a TG exon-7 highly mutation-prone region, and the possibility that some cases were introduced to South America from Galicia cannot be excluded.
Assuntos
Hipotireoidismo Congênito/genética , Bócio/genética , Tireoglobulina/genética , Adulto , Western Blotting , Células Cultivadas , Testes Genéticos , Haplótipos , Humanos , Imunoprecipitação , Masculino , Microscopia Eletrônica , Mutação/genética , Linhagem , EspanhaRESUMO
OBJECTIVE: Turner syndrome (TS) is an indication for GH therapy in spite of the modest growth response. Somatic growth depends not only on GH insulin-like growth factor I (IGF-I) axis but also on thyroid hormone (TH) status. We have previously reported that supraphysiological IGF-I levels diminished TH actions in rat tissues by reducing the nuclear TH receptor (TR). GH treatment to TS patients induces high IGF-I levels and therefore a reduction of TH action in tissues may be expected. We aimed at evaluating the effect of GH therapy in TS girls on peripheral TH action. DESIGN AND PATIENTS: We set up a reverse transcription-polymerase chain reaction (RT-PCR) for TR mRNA estimation in peripheral blood mononuclear cells (PBMC) and compared TR mRNA levels from 10 normal, 10 TS and 10 TS girls under GH therapy (0.33 mg/kg/week for 0.5-2 years). MEASUREMENTS: After RNA extraction from PBMC, TR and beta-actin mRNAs were coamplified by RT-PCR. In addition serum biochemical markers of TH action were measured: thyrotropin (TSH), sex hormone binding globulin (SHBG), osteocalcin (OC), beta-crosslaps (beta-CL), iodothyronines by electrochemiluminescency and IGF-I by immunoradiometric assay (IRMA) with extraction. RESULTS: TR mRNAs from PBMC were reduced in TS patients under GH treatment. In turn, serum TSH, OC, beta-CL and IGF-I were increased while SHBG was reduced by GH treatment in TS patients. CONCLUSIONS: GH treatment reduced TR expression in PBMC and biochemical serum markers of TH action. These results suggest that GH treatment in TS patients impair peripheral TH action at tissue level and prompt a role in the reduced growth response to the therapy.
Assuntos
Hormônio do Crescimento/uso terapêutico , Hormônios Tireóideos/sangue , Síndrome de Turner/tratamento farmacológico , Actinas/genética , Análise de Variância , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Colágeno/sangue , Depressão Química , Feminino , Seguimentos , Humanos , Fator de Crescimento Insulin-Like I/análise , Osteocalcina/sangue , Fragmentos de Peptídeos/sangue , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Globulina de Ligação a Hormônio Sexual/análise , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Tireotropina/sangue , Tri-Iodotironina/sangue , Síndrome de Turner/sangueRESUMO
Las enfermedades tiroideas constituyen una heterogénea colección de anormalidades asociadas a mutaciones en los genes responsables en el desarrollo de la tiroides: factor de transcripción tiroideo 1 (TTF-1), factor de transcripción tiroideo 2 (TTF-2) y PAX8, o en uno de los genes que codifican para las proteínas involucradas en la biosíntesis de hormonas tiroideas como tiroglobulina (TG), tiroperoxidasa (TPO),sistema de generación de peróxido de hidrógeno (DUOX2), cotransportdor de Na/I (NIS), pendrina (PDS), TSH y receptor de TSH. El hipotiroidismo congénito ocurre con una prevalencia de 1 en 4.000 nacidos. Los pacientes coneste síndrome pueden ser divididos en dos grupos: con hipotiroidismo congénito sin bocio (disembriogénesis) o con bocio (dishormonogénesis). El grupo de disembriogénesis, que corresponde al 85% de los casos, resulta de ectopía,agenesia o hipoplasia. En una minoría de estos pacientes, el hipotiroidismo congénito está asociado con mutaciones en los genes TTF-1, TTF-2, PAX-8, TSH o TSHr. La resencia de bocio congénito (15% de los casos) se ha asociado a mutaciones en los genes NIS, TG, TPO, DUOX2 o PDS. El hipotiroidismo congénito por dishormonogénesis es trasmitido en forma autonómica recesiva. Mutaciones somáticas en el TSHr han sido identificadas en adenomas tiroideos hiperfuncionantes. Otra enfermedad tiroidea bien establecida es la resistencia a hormonas tiroideas(RTH). Es un síndrome de reducida respuesta tisular a la acción hormonal causado por mutaciones localizadas en el gen del receptor de hormonas tiroideas (TR). Mutantes de TRinterfieren con la función del receptor normal por un mecanismo de dominancia negativa. En conclusión, la identificación de mutaciones en los genes de expresión tiroidea ha permitido un mayor entendimiento sobre la relación estructura-función de los mismos. La tiroides constituye un excelente modelo para el estudio molecular de las enfermedades genéticas.
Assuntos
Humanos , Bócio/genética , Hipertireoidismo/genética , Hipotireoidismo/genética , Bócio/metabolismo , Hipertireoidismo/metabolismo , Hipotireoidismo/congênito , Hipotireoidismo/metabolismo , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Mutação , Receptores da Tireotropina/genética , Hormônios Tireóideos/biossíntese , Hormônios Tireóideos/genéticaRESUMO
La diabetes autoinmune es una enfermedad multifactorial causada por factores genéticos predisponentes y ambientales desencadenantes. Se manifiesta en la edad infantojuvenil (diabetes tipo 1, DMID) y en la edad adulta (diabetes autoinmune latente del adulto, LADA). La predisposición genética es de tipo poligénico, se ha establecido asociación con alelos polimórficos del gen DQB del sistema HLA, VNTR del gen de insulina y polimorfismos en el gen CTLA4. En el presente trabajo se analizaron las frecuencias de los alelos polimórficos del gen HLA DQB1 en 63 pacientes LADA, 70 pacientes DMID y 79 individuos normales. La tipificación de los alelos del gen DQB1 se llevó a cabo mediante el Kit SSPTM DQ Olerup. Se observó una mayor frecuencia del genotipo *0201-*0302 y *0201-*0201 en ambas poblaciones diabéticas con respecto a normales (p<0.05). La presencia del genotipo *0201-*0302 fue mayor en DMID que en LADA (p<0.05). Por otra parte, el análisis del alelo protector *0602 muestra una alta prevalencia en individuos normales con respecto a la población diabética. El alelo de susceptibilidad más frecuente en pacientes LADA y DMID de nuestro país fue el *0201. En conclusión, LADA presenta susceptibilidad genética dada por alelos del gen HLA DQB1 pero en forma menos determinante que en diabetes tipo 1. A su vez, el hallazgo del aumento en la frecuencia del alelo *0201, tanto en frecuencias alélicas como genotípicas permite caracterizar nuestra población de pacientes tanto LADA como DMID a diferencia de otras poblaciones en las que el alelo más frecuente es el *0302.
Assuntos
Adulto , Humanos , Doenças Autoimunes/genética , Diabetes Mellitus Tipo 1/genética , Genótipo , Frequência do Gene/genética , Antígenos HLA-DQ/genética , Polimorfismo Genético/genética , Idade de Início , Argentina , Estudos de Casos e Controles , Razão de ChancesRESUMO
Las enfermedades tiroideas constituyen una heterogénea colección de anormalidades asociadas a mutaciones en los genes responsables en el desarrollo de la tiroides: factor de transcripción tiroideo 1 (TTF-1), factor de transcripción tiroideo 2 (TTF-2) y PAX8, o en uno de los genes que codifican para las proteínas involucradas en la biosíntesis de hormonas tiroideas como tiroglobulina (TG), tiroperoxidasa (TPO),sistema de generación de peróxido de hidrógeno (DUOX2), cotransportdor de Na/I¹ (NIS), pendrina (PDS), TSH y receptor de TSH. El hipotiroidismo congénito ocurre con una prevalencia de 1 en 4.000 nacidos. Los pacientes coneste síndrome pueden ser divididos en dos grupos: con hipotiroidismo congénito sin bocio (disembriogénesis) o con bocio (dishormonogénesis). El grupo de disembriogénesis, que corresponde al 85% de los casos, resulta de ectopía,agenesia o hipoplasia. En una minoría de estos pacientes, el hipotiroidismo congénito está asociado con mutaciones en los genes TTF-1, TTF-2, PAX-8, TSH o TSHr. La resencia de bocio congénito (15% de los casos) se ha asociado a mutaciones en los genes NIS, TG, TPO, DUOX2 o PDS. El hipotiroidismo congénito por dishormonogénesis es trasmitido en forma autonómica recesiva. Mutaciones somáticas en el TSHr han sido identificadas en adenomas tiroideos hiperfuncionantes. Otra enfermedad tiroidea bien establecida es la resistencia a hormonas tiroideas(RTH). Es un síndrome de reducida respuesta tisular a la acción hormonal causado por mutaciones localizadas en el gen del receptor de hormonas tiroideas (TR). Mutantes de TRinterfieren con la función del receptor normal por un mecanismo de dominancia negativa. En conclusión, la identificación de mutaciones en los genes de expresión tiroidea ha permitido un mayor entendimiento sobre la relación estructura-función de los mismos. La tiroides constituye un excelente modelo para el estudio molecular de las enfermedades genéticas. (AU)
Assuntos
Humanos , Hipertireoidismo/genética , Bócio/genética , Hipotireoidismo/genética , Hipertireoidismo/metabolismo , Bócio/metabolismo , Mutação , Hormônios Tireóideos/biossíntese , Hormônios Tireóideos/genética , Receptores da Tireotropina/genética , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Hipotireoidismo/metabolismo , Hipotireoidismo/congênitoRESUMO
La diabetes autoinmune es una enfermedad multifactorial causada por factores genéticos predisponentes y ambientales desencadenantes. Se manifiesta en la edad infantojuvenil (diabetes tipo 1, DMID) y en la edad adulta (diabetes autoinmune latente del adulto, LADA). La predisposición genética es de tipo poligénico, se ha establecido asociación con alelos polimórficos del gen DQB del sistema HLA, VNTR del gen de insulina y polimorfismos en el gen CTLA4. En el presente trabajo se analizaron las frecuencias de los alelos polimórficos del gen HLA DQB1 en 63 pacientes LADA, 70 pacientes DMID y 79 individuos normales. La tipificación de los alelos del gen DQB1 se llevó a cabo mediante el Kit SSPTM DQ Olerup. Se observó una mayor frecuencia del genotipo *0201-*0302 y *0201-*0201 en ambas poblaciones diabéticas con respecto a normales (p<0.05). La presencia del genotipo *0201-*0302 fue mayor en DMID que en LADA (p<0.05). Por otra parte, el análisis del alelo protector *0602 muestra una alta prevalencia en individuos normales con respecto a la población diabética. El alelo de susceptibilidad más frecuente en pacientes LADA y DMID de nuestro país fue el *0201. En conclusión, LADA presenta susceptibilidad genética dada por alelos del gen HLA DQB1 pero en forma menos determinante que en diabetes tipo 1. A su vez, el hallazgo del aumento en la frecuencia del alelo *0201, tanto en frecuencias alélicas como genotípicas permite caracterizar nuestra población de pacientes tanto LADA como DMID a diferencia de otras poblaciones en las que el alelo más frecuente es el *0302. (AU)
Assuntos
Adulto , Humanos , RESEARCH SUPPORT, NON-U.S. GOVT , Antígenos HLA-DQ/genética , Genótipo , Doenças Autoimunes/genética , Diabetes Mellitus Tipo 1/genética , Polimorfismo Genético/genética , Frequência do Gene/genética , Estudos de Casos e Controles , Razão de Chances , Idade de Início , ArgentinaRESUMO
OBJECTIVE: In order to complete the knowledge of the genomic organization of the human thyroglobulin gene, the present work was designed to establish the intron-exon organization from exon 24 to exon 35 and to construct a more complete physical map of the gene. DESIGN: Screening of two genomic libraries, and subsequent restriction mapping, hybridization and sequencing were used to characterize the recombinant phages. METHODS: Two human genomic DNA libraries were screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. The Long PCR method was used to amplify the genomic DNA region containing exon 24. Intron-exon junction sequences were determined by using the Taq polymerase-based chain termination method. RESULTS: We isolated and characterized five lambda phage clones that include nucleotides 4933 to 6262 of the thyroglobulin mRNA, encompassing exons 25-35 of the gene. The remaining exon 24 (nucleotides 4817-4932) was sequenced from the amplified fragment. In total, 8010 intronic bases were analyzed. CONCLUSIONS: The present study shows that the five phages isolated and the amplified fragment include 59.4 kb genomic DNA, covering 1446 nucleotides of exonic sequence distributed over 12 exons, from exon 24 to exon 35. Using previous studies and our current data, 220 kb of the human thyroglobulin gene was analyzed, a physical map was constructed, and all exon-intron junctions were sequenced and correlated with the different domains of the protein. In summary, the thyroglobulin gene contains 48 exons ranging in size from 63 nucleotides to 1101 nucleotides.
Assuntos
Éxons , Íntrons , Tireoglobulina/genética , Sequência de Bases/genética , Mapeamento Cromossômico , DNA/genética , Genoma , Humanos , Dados de Sequência MolecularRESUMO
In this work we have extended our initial molecular studies of a consanguineous family with two affected goitrous siblings (H.S.N. and Ac.S.N.) with defective thyroglobulin (Tg) synthesis and secretion because of a homozygotic deletion of a fragment of 138 nucleotides (nt) in the central region of the Tg mRNA, identified previously in H.S.N. In order to identify the intron/exon boundaries and to analyze the regions responsible for pre-mRNA processing corresponding to a 138 nt deletion, we performed a screening of a human genomic library. The intron/exon junction sequences were determined from one positive clone by sequencing both strands of the DNA template. The results showed that the deletion mapped between positions 5549 and 5686 of the Tg mRNA and corresponded to exon 30. The positions of the exon limits differed by three nucleotides from the previously reported data obtained from direct sequencing of the deleted reverse transcriptase-polymerase chain reaction fragment from H.S.N. These variations are because the intron/exon junctions in this region were not available at the time when the deletion was first described. The deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shortened by 46 residues. The same 138 nt deletion was observed in reverse transcriptase-polymerase chain reaction studies performed in the thyroid tissues from Ac.S.N. Genomic DNA analysis showed that a G to T transversion was observed at position +1 in the donor site of intron 30. Both affected patients (H.S.N. and Ac.S.N.) are homozygous for the mutation whereas the normal sister (At.S.N.) had a normal allele pattern. The functional consequences of the deletion are related to structural changes in the protein molecule that either could modify the normal routing of the translation product through the membrane system of the cell or could impair the coupling reaction. Probably the mutant Tg polypeptide might be functionally active in the production of thyroid hormone, because in the presence of a normal iodine ingestion (approximately 150 microg/day), Ac.S.N. was able to maintain normal serum levels of total triiodothyronine (T3) associated with relatively low serum total thyroxine (T4) with normal somatic development without signs of brain damage.
Assuntos
DNA Recombinante , Bócio/congênito , Bócio/genética , Hipotireoidismo/genética , Mutação/fisiologia , Tireoglobulina/genética , Adulto , Sequência de Aminoácidos/genética , Sequência de Bases/genética , DNA Complementar/genética , Éxons/genética , Genoma , Humanos , Íntrons/genética , Masculino , Dados de Sequência Molecular , Mutação/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The coding region of the human thyroglobulin (TG) mRNA has been resequenced, and comparison with the TG sequence originally published in 1987 showed many variations. All of the variations were validated in 20--40 other alleles, and this resulted in the revision of 41 nucleotide positions. This review presents the revised wild-type human TG sequence, including all known exon/exon boundaries and additional data on the TG mRNA population, concerning alternative splicing and variability of the polyadenylation cleavage site. The amino acid sequence derived shows one additional, 12 changed, and 10 polymorphic residues. Protein characteristics, such as acceptor and donor tyrosine residues, N-glycosylation sites, cysteine-rich repeats, the proposed receptor domain, and antigenic epitopes, are included, and their relationship to the revised sequence is discussed. Furthermore, all reported TG mutations causing dyshormonogenesis in humans and animals are designated in the nucleotide and amino acid sequences. This up-to-date profile of the human TG molecule presents the features of importance for its complex role in thyroid hormonogenesis, and is the basis for future studies on the structure--function relationship.
Assuntos
RNA Mensageiro/análise , Tireoglobulina/genética , Processamento Alternativo , Sequência de Aminoácidos , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Sequência de Bases , Sequência Conservada , Epitopos , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Análise de Sequência de DNA , Tireoglobulina/biossíntese , Doenças da Glândula Tireoide/genética , Doenças da Glândula Tireoide/metabolismo , Glândula Tireoide/metabolismoRESUMO
OBJECTIVE: The purpose of the present work is to establish the intron-exon organization from exon 12 to exon 23 of the human thyroglobulin gene and to construct a physical map of the 5' terminal half of the gene. DESIGN: Screening of a genomic library and subsequent restriction map, hybridization and sequencing methods have been employed to characterize the recombinant positive phages. METHODS: A human genomic DNA library was screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. Intron/exon junction sequences were determined by the Taq polymerase-based chain terminator method. Finally, the thyroglobulin gene was mapped using the Gene Bridge 4 radiation hybrid clone panel. RESULTS: We isolated and characterized four lambda phage clones that include nucleotides 3002 to 4816 of the thyroglobulin mRNA, encompassing exons 12 to 23 of the gene. The exon sizes range between 78 and 219 nucleotides. We found that the GT-AG splicing sequences rule was perfectly respected in all the introns. A total of 7302 intronic bases was analyzed. Hormogenic tyrosine 5 and 1291 are encoded by exons 2 and 18. Also, seven alternative spliced variants are associated with the 5' region. Thyroglobulin gene maps to 5,5 centiRays from the AFMA053XF1 marker, in chromosome 8. CONCLUSIONS: The present study shows that the first 4857 bases of thyroglobulin mRNA are divided into 23 exons and the four phages isolated include 32.6 kb genomic DNA, covering 1815 nucleotides of exonic sequence distributed in 12 exons, from exon 12 to 23.
Assuntos
Regiões 5' não Traduzidas/genética , Tireoglobulina/genética , Sequência de Bases , Primers do DNA , Éxons , Biblioteca Genômica , Humanos , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Mapeamento por RestriçãoAssuntos
Doenças da Glândula Tireoide/genética , Hormônios Tireóideos/biossíntese , Humanos , Hipotireoidismo/genética , Recém-Nascido , Iodeto Peroxidase/genética , Tireoglobulina/genética , Tireoglobulina/metabolismo , Glândula Tireoide/fisiologia , Hormônios Tireóideos/genética , Tireotropina/metabolismoAssuntos
Humanos , Lactente , Doenças da Glândula Tireoide/genética , Hormônios Tireóideos/biossíntese , Tireoglobulina/genética , Tireoglobulina/metabolismo , Glândula Tireoide/fisiologia , Hormônios Tireóideos/genética , Tireotropina/metabolismo , Hipotireoidismo/genética , Iodeto Peroxidase/genéticaRESUMO
The genomic organization of the 3' end of the human Thyroglobulin (Tg) gene has not previously been characterized. We isolated and characterized seventeen lambda phage clones from a human genomic library that included nucleotides 6263 to 8410 of the Tg mRNA, encompassing the last thirteen 3' exons of the Tg gene. The region contained exons ranging in size from 94 to 222 nucleotides, split by introns of 1 to 64 kb. We estimate a total of 48 exons in the Tg gene. All the intron-exon boundaries were sequenced. We found that the splicing sequences diverged considerably from the 3' and 5' consensus. However, the GT-AG rule was perfectly respected in all the exons. A total of 5788 intronic bases and most of the sequences contained in the 13 exons were analyzed (1846 bases). One sequence variation, TT to CC at positions 8377-8378, was found in the 3' untranslated segment. The three tyrosine residues involved in thyroid hormones synthesis (amino acids 2554, 2568, and 2747) at the carbosyl termini of Tg, are encoded by exons 44, 45, and 48. The knowledge of the precise organization of the Tg gene should help to direct studies of Tg gene mutations in families in which a defect in the synthesis of Tg occurs.
Assuntos
Genoma Humano , Tireoglobulina/genética , Bacteriófago lambda/genética , Sequência de Bases , DNA/genética , Primers do DNA/genética , Éxons , Variação Genética , Humanos , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , RNA Mensageiro/genéticaRESUMO
PURPOSE: Was to establish the molecular genetics of thalassemias in Argentina. PATIENTS AND METHODS: Genomic DNA was amplified by PCR and six point mutations in the beta-globin gene were investigated by Dot Blot hybridization using oligonucleotide probes. The most frequent alpha-thalassemia deletions were studied by Southern Blotting. Patients were distributed in 4 groups: a) 109 beta-thalassemic carriers; b) 15 thalassemia major patients; c) 2 thalassemia intermedia patients and d) 14 probable alpha-thalassemic carriers. RESULTS: The distribution of mutated alleles in the group a) was: IVS-1 nt 1: 13.76%, IVS-1 nt 6: 7.34%, IVS-1 nt 110: 23.85%, codon 39: 39.45%, IVS-2 nt 1: 3.68% e IVS-2 nt 745: 1.83%, 10.01% could not be determined with the probes used; in the group b) the allelic distribution was similar and the compound genetic genotype were predominant related to homocygous ones; in the group c): we confirmed the presence of one beta-thalassemia mutation and a alpha gene triplication (alpha alpha alpha) in the 2 patients studied. The alpha-thalassemia character was confirmed in 8 patients of the group d) (6 had -alpha 3,7/alpha alpha genotype and 2,-alpha 3,7/-alpha 3,7 genotype). CONCLUSIONS: This study indicates that the analysis of 6 mutations in the beta-globin gene and the alpha-globin gene deletions are an effective strategy to identify thalassemias in Argentina.
Assuntos
Globinas/genética , Mutação Puntual , Talassemia/genética , Alelos , Argentina/epidemiologia , Códon/genética , Análise Mutacional de DNA , Frequência do Gene , Genótipo , Humanos , Immunoblotting , Reação em Cadeia da Polimerase , Deleção de Sequência , Talassemia/epidemiologiaRESUMO
Impaired thyroglobulin (Tg) synthesis is one of the putative causes for dyshormonogenesis of the thyroid gland. This type of hypothyroidism is characterized by intact iodide trapping, normal organification of iodide, and usually low serum Tg levels in relation to high TSH, and when untreated the patients develop goiter. In thyroid tissue from a 13-yr-old patient suspected of a thyroglobulin synthesis defect, the Tg mRNA was studied. The complete coding region of 8307 bp was directly sequenced and revealed a homozygous point mutation: a C886T transition in exon 7. Upon translation this mutation would result in a stopcodon at amino acid position 277, replacing the arginine residue. A Tg cDNA construct containing the mutation was expressed in rabbit reticulocyte lysate resulting in a truncated protein of 30 kDa. Expression in the presence of microsomal membranes resulted in a gel shift of this Tg molecule, indicating glycosylation ability. Two other siblings had a clinical presentation like the index patient, while their parents were unaffected. Additional restriction fragment length polymorphism analysis of the pedigree verified that the homozygous nonsense mutation cosegregated with the clinical phenotype. Clinically, hypothyroidism was not severe in the affected siblings because the truncated Tg glycoprotein was still capable of thyroid hormonogenesis.
Assuntos
Códon , Bócio/genética , Hipotireoidismo/genética , Mutação , RNA Mensageiro/química , Tireoglobulina/genética , Adolescente , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Pré-Escolar , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Tireoglobulina/químicaRESUMO
We have previously reported a Brazilian family with congenital goiter, hypothyroidism, and marked impairment of thyroglobulin (Tg) synthesis. Analysis of the Tg mRNA in the goiter of one of the siblings revealed a cytosine to thymine transition creating a stop codon at position 1510. This point mutation is removed from the majority of Tg mRNA transcripts by the preferential generation in the goiter of a 171 nt deleted Tg mRNA by alternative splicing. The nonsense mutation destroys a TaqI site at this position in the mutant Tg gene. Using polymerase chain reaction (PCR) amplification and TaqI digestion we found that two siblings affected with goiter and hypothyroidism, as well as the father and three siblings with normal thyroid function, are all heterozygous for the nonsense mutation. This implies that an additional mutation must be present in the affected individuals, generating a compound heterozygote genotype. A new polymorphism within the thyroglobulin gene represented by three alleles has been detected. This was documented by the TaqI restriction enzyme and phTgM3 probe hybridization that showed a three allelic polymorphism with fragment sizes of 16.5 kb (allele A), 14.5 kb (allele B) and 11.0 kb (allele C). Segregation analysis of these alleles in the family indicated that the two affected siblings were homozygous for the allele C. In contrast the unaffected father and three other siblings, who carried the nonsense mutation, were heterozygous for alleles B and C. Analysis of the Tg genotypes implies that two additional mutations of the Tg gene must segregate in this family to account for the observed phenotypes.
Assuntos
Bócio/genética , Hipotireoidismo/genética , Mutação/fisiologia , Tireoglobulina/genética , Adulto , Alelos , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Brasil , Códon sem Sentido/genética , Hipotireoidismo Congênito , DNA/análise , DNA/genética , Feminino , Frequência do Gene , Genoma Humano , Bócio/congênito , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da PolimeraseRESUMO
We analyzed thyroglobulin (Tg) reverse transcription polymerase chain reaction (RT-PCR) products from three congenital goiters and three normal thyroid tissues by Taq I digestion. Tg coding sequences were amplified from position 57 to 8448 in 12 amplification fragments. A Taq I restriction fragment length polymorphism was detected in the most 3' RT-PCR product (nt 7584 through 8448). Data from the sequence showed a G-->A transition (nt 7627) causing the disappearance of the Taq I site in position 7625. It produced the substitution of arginine for a glutamine at position 2510. Afterwards, we established that the glutamine allele is present in normal unrelated individuals, with an allelic frequency of 62%. This Tg variant is thus widely represented in the human population. The available sequence information from rat and bovine Tg showed the presence, in both, of glutamine at position 2510.
