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Several patients were identified with dyshormonogenesis caused by mutations in the thyroglobulin (TG) gene. These defects are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous for the mutations. The aim of the present study was to identify new TG mutations in a patient of Vietnamese origin affected by congenital hypothyroidism, goiter and low levels of serum TG. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the maternal mutation consists of a novel c.745+1G>A (g.IVS6 + 1G>A), whereas the hypothetical paternal mutation consists of a novel c.7036+2T>A (g.IVS40 + 2T>A). The father was not available for segregation analysis. Ex-vivo splicing assays and subsequent RT-PCR analyses were performed on mRNA isolated from the eukaryotic-cells transfected with normal and mutant expression vectors. Minigene analysis of the c.745+1G>A mutant showed that the exon 6 is skipped during pre-mRNA splicing or partially included by use of a cryptic 5' splice site located to 55 nucleotides upstream of the authentic exon 6/intron 6 junction site. The functional analysis of c.7036+2T>A mutation showed a complete skipping of exon 40. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses predicted that both mutant alleles would result in the abolition of the authentic splice donor sites. The c.745+1G>A mutation originates two putative truncated proteins of 200 and 1142 amino acids, whereas c.7036+2T>A mutation results in a putative truncated protein of 2277 amino acids. In conclusion, we show that the c.745+1G>A mutation promotes the activation of a new cryptic donor splice site in the exon 6 of the TG gene. The functional consequences of these mutations could be structural changes in the protein molecule that alter the biosynthesis of thyroid hormones., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The objective of this study was to perform genetic analysis in three brothers of Turkish origin born from consanguineus parents and affected by congenital hypothyroidism, goiter and low levels of serum TG. The combination of sequencing of DNA, PCR mapping, quantitative real-time PCR, inverse-PCR (I-PCR), multiplex PCR and bioinformatics analysis were used in order to detect TG mutations. We demonstrated that the three affected siblings are homozygous for a DNA inversion of 16,962bp in the TG gene associated with two deleted regions at both sides of the inversion limits. The inversion region includes the first 9bp of exon 48, 1015bp of intron 47, 191bp of exon 47, 1523bp of intron 46, 135bp of exon 46 and the last 14,089bp of intron 45. The proximal deletion corresponds to 27bp of TG intron 45, while the distal deletion spans the last 230bp of TG exon 48 and the first 588bp of intergenic region downstream TG end. The parents were heterozygous carriers of the complex rearrangement. In conclusion, a novel large imperfect DNA inversion within the TG gene was identified by the strategy of I-PCR. This aberration was not detectable by normal sequencing of the exons and exon/intron boundaries. Remarkably, the finding represents the first description of a TG deficiency disease caused by a DNA inversion., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The thyroglobulin (TG) gene is organized in 48 exons, spanning over 270 kb on human chromosome 8q24. Up to now, 62 inactivating mutations in the TG gene have been identified in patients with congenital goiter and endemic or non-endemic simple goiter. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report 13 patients from seven unrelated families with goiter, hypothyroidism and low levels of serum TG. All patients underwent clinical, biochemical and imaging evaluation. Single-strand conformation polymorphism (SSCP) analysis, endonuclease restriction analysis, sequencing of DNA, genotyping, population screening, and bioinformatics studies were performed. Molecular analyses revealed seven novel inactivating TG mutations: c.378C>A [p.Y107X], c.2359C>T [p.R768X], c.2736delG [p.R893fsX946], c.3842G>A [p.C1262Y], c.5466delA [p.K1803fsX1833], c.6000C>G [p.C1981W] and c.6605C>G [p.P2183R] and three previously reported mutations: c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.7006C>T [p.R2317X]. Six patients from two families were homozygous for p.R277X mutation, four were compound heterozygous mutations (p.Y107X/p.C1262Y, p.R893fsX946/p.A2215D, p.K1803fsX1832/p.R2317X), one carried three identified mutations (p.R277X/p.C1981W-p.P2183R) together with a hypothetical micro deletion and the remaining two siblings from another family with typical phenotype had a single p.R768X mutated allele. In conclusion, our results confirm the genetic heterogeneity of TG defects and the pathophysiological importance of altered TG folding as a consequency of truncated TG proteins and missense mutations located in ACHE-like domain or that replace cysteine., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Thyroglobulin (TG) is a homodimeric glycoprotein synthesized by the thyroid gland. To date, 52 mutations of the TG gene have been identified in humans. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report a French patient with congenital hypothyroidism, mild enlarged thyroid gland and low levels of serum TG. Sequencing of DNA, genotyping, expression of chimeric minigenes as well as bioinformatics analysis were performed. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the paternal mutation consists of a c.3788-3789insT or c.3788dupT, whereas the maternal mutation consists of g.IVS19+3_+4delAT. Minigene analysis of the g.IVS19+3_+4delAT mutant showed that the exon 19 is skipped during pre-mRNA splicing or partially included by use of cryptic 5' splice site located to 100 nucleotides downstream of the wild type exon-intron junction. The c.3788-3789insT mutation results in a putative truncated protein of 1245 amino acids, whereas g.IVS19+3_4delAT mutation originates two putative truncated proteins of 1330 and 1349 amino acids. In conclusion, we show that the g.IVS19+3_+4delAT mutation promotes the activation of a cryptic donor splice site in the exon 19 of the TG gene. These results open up new perspectives in the knowledge of the mechanism of splicing for the TG pre-mRNA., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The autoimmune thyroid disease is a complex disorder caused by a combination of genetic susceptibility and environmental factors, which are believed to initiate the autoimmune response to thyroid antigens. Identification of the susceptibility genes has found that unique and diverse genetic factors are in association with Graves' disease and autoimmune thyroiditis. The thyroglobulin gene is an identified thyroid-specific gene associated to autoimmune thyroid disease and, principally, with autoimmune thyroiditis. The aim of this work was to test for evidence of allelic association between autoimmune thyroiditis and thyroglobulin polymorphism markers. We studied six polymorphisms distributed throughout all the thyroglobulin gene: four microsatellites (Tgms1, Tgms2, TGrI29 and TGrI30), one insertion/deletion (Indel) polymorphism (IndelTG-IVS18) and one exonic single nucleotide polymorphism (SNP) (c.7589G>A) in 122 patients with autoimmune thyroiditis compared with 100 non-related normal subjects. No differences in allele and genotype distribution were observed between autoimmune thyroiditis cases and controls for Tgms1, Tgms2, TGrI30, IndelTG-IVS18 and c.7589G>A. However, when we analyzed the patients with the TGrI29 microsatellite we found a significant association between the 199-bp allele and AT (33.7% vs. 24.5% in control group) (P = 0.0372). In addition, a higher prevalence of the 201-bp allele has been observed in control subjects (47.5% vs. 38.1% in patients group), although not statistically significant (P = 0.0536). Our work shows the association between the thyroglobulin gene and autoimmune thyroiditis and reinforce that thyroglobulin is a thyroid-specific susceptibility gene for this disease.

Background: Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre., Objectives: The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied., Methods: Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed., Results: Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing., Conclusions: In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.

Thyroglobulin (TG) functions as the matrix for thyroid hormone synthesis. Thirty-five different loss-of-function mutations in the TG gene have been reported. These mutations are transmitted in an autosomal recessive mode. The objective of this study is to analyze the recurrence of the p.R277X/p.R1511X compound heterozygous mutation in the TG gene in two unrelated families (one Argentinian and another Brazilian) with congenital hypothyroidism, goiter and impairment of TG synthesis. The first and last exon of the TG gene, the exons where previously mutations and single nucleotide polymorphisms (SNPs) were detected, as well as the TG promoter, were analyzed by automatic sequencing in one affected member of the each family. Four microsatellite markers localized in introns 10, 27, 29 and 30 of the TG gene, one insertion/deletion intragenic polymorphism and 15 exonic SNPs were used for haplotype analysis. A p.R277X/p.R1511 compound heterozygous mutation in the TG gene was found in two members of an Argentinian family. The same mutations had been also reported previously in two members of a Brazilian family. We constructed mutation-associated haplotypes by genotyping members of the two families. Our results suggest that the cosegregating haplotype is different in each one of these families. Different haplotypes segregated with the p.R277X and p.R1511 mutations demonstrating the absence of a founder effect for these mutations between Argentinian and Brazilian populations. However, haplotyping of Argentinian patients showed the possibility that the p.R277X alleles might be derived from a common ancestral chromosome.

Context: Thyroid dyshormonogenesis is associated with mutations in the thyroglobulin (TG) gene and characterized by normal organification of iodide and low serum TG. These mutations give rise to congenital goitrous hypothyroidism, transmitted in an autosomal recessive mode., Objectives: The aim of this study was to identify new mutations in the TG gene in an attempt to increase the understanding of the molecular basis of this disorder. Three unrelated patients with marked impairment of TG synthesis were studied., Methods: The promoter and the complete coding regions of the TG gene, along with the flanking intronic regions, were analysed by direct DNA sequencing., Results: Four different inactivating TG mutations, three novel mutations (c.548G>A, p.C164Y; c.759-760insA, p.L234fsX237; c.6701C>A, p.A2215D) and one previously identified mutation (c.886C>T, p.R277X) were identified. Multiple sequence alignment study revealed that the wild-type cysteine residue at position 164 is strictly conserved in the TG of all the species analysed, whereas the wild-type alanine residue at position 2215 is well conserved in the TG and acetylcholinesterase (AChE) of all the species analysed except in rabbit AChE, in which it is substituted by glutamic acid., Conclusions: We report three patients with congenital hypothyroidism with goitre caused by two compound heterozygous mutations, p.C164Y/p.L234fsX237 and p.R277X/p.A2215D, and one homozygous mutation, p.R277X, in the TG gene. To our knowledge this is the first report of the presence of a nucleotide insertion mutation in the TG gene.

Identification of thyroglobulin (TG) gene mutations may provide insight into the structure-function relationship. In this study, we have performed molecular studies in a patient with congenital goiter, hypothyroidism, and impairment of TG synthesis. Genomic DNA sequencing revealed a homozygous c.886C-->T mutation in exon 7, resulting in a premature stop codon at amino acid 277 (p.R277X). The same nonsense mutation had been reported previously in two Brazilian families with multiple occurrence of congenital hypothyroidism with goiter. We compared the insertion/deletion polymorphism in intron 18, microsatellites (Tgm1, Tgm2, TGrI29, and TGrI30), and exonic single-nucleotide polymorphism haplotypes identified in the patient with a member of the previously reported family, who also carry the mutation as a compound heterozygous mutation. The single-nucleotide polymorphism and microsatellite analysis revealed that the two affected individuals do not share a common TG allele. This suggests that the p.R277X mutation is a mutational hot spot. No difference in either splicing or abundance of the amplified product was detected by RT-PCR, excluding that an alternative splicing mechanism, by skipping of exon 7, would restore the normal reading frame. In conclusion, we report a new case of congenital goiter and hypothyroidism caused by a p.R277X mutation in the TG gene. Moreover, we show that nucleotide 886 is a mutational hot spot that explains the recurrence of this mutation.

Introduction: We have described in previous articles a nonsense mutation (4588C>T, R1511X) in exon 22 of the thyroglobulin (TG) gene in a member of a family with a complex history of congenital goiter. In the mutated thyroid gland, full-length thyroglobulin mRNA is almost undetectable. However, a smaller transcript is detected in which the mutated exon 22 is skipped and the reading frame restored. It is conceivable that alternative splicing might be a mechanism involved in the rescue of nonsense mutations., Methods: To investigate whether the detection of the alternative mRNA is due to an increase in its concentration or its preferential amplification during reverse transcriptase-PCR in the absence of the normal full-length mRNA competitor, we set up an assay in which the competitor mRNA was provided. We also studied the effect of the 4588C>T mutation on exon definition and processing using wild-type and mutated minigenes., Results: The detection of the alternative mRNA lacking exon 22 is not caused by the absence of the full-length competitor. In contrast, our results demonstrate that the alternative transcript preferentially accumulates in the mutated thyroid at a level similar to the full-length transcript in control tissue. Transient expression experiments with wild-type and mutated minigenes indicate that the mutated exon is as efficiently spliced as the wild-type, suggesting that the 4588C>T mutation does not interfere with exon 22 definition and processing., Conclusions: The alternative splicing of the TG gene described in this article constitutes a new case of nonsense-associated alternative splicing. We have shown that the mutation itself does not interfere with exon definition and processing in vitro. Our results support the hypothesis that the alternative splicing of the mutated exon is driven by the interruption of the reading frame.

The cause of sporadic simple goiter is unknown in most cases. Family studies have suggested that this disorder may have a genetic component in some patients. We have previously demonstrated that some cases of endemic and nonendemic simple goiter are associated with a mutation within exon 10 of the thyroglobulin gene. Here we report a study of 50 cases diagnosed as having nonendemic simple goiter, and found 1 case with a large heterozygous deletion within the thyroglobulin gene. The deletion involves the promoter region and the 11 first exons of this gene and is associated with a euthyroid state. We hypothesize that the absence of thyroglobulin synthesis from the deleted allele may be responsible for a decreased level of thyroglobulin mRNA. Euthyroidism would be achieved by thyrotropin (TSH) stimulation but at the expense of goiter development.

Iodine deficiency is the most relevant etiologic factor in endemic goiter. However, the fact that not all residents in the same area eventually develop goiter suggests that individual factors might also be involved in the etiology of endemic goiter. We have previously reported a point mutation in thyroglobulin exon 10 associated with nonendemic simple goiter. In an attempt to determine whether the mutation in thyroglobulin exon 10 might be linked to endemic goiter, we studied the genomic organization of thyroglobulin exon 10 in 36 patients diagnosed with endemic goiter by Southern blot, PCR, and sequencing analysis. We also analyzed by Southern blot the organization of the genomic region that contains thyroglobulin exons 1 to 11. In one case, we observed a point mutation in thyroglobulin exon 10. Sequencing analysis revealed a mutation at position 2610 of the cDNA, which implies a G to T substitution. This single base change results in a glutamine to histidine substitution and is the same as that previously reported by our group in patients with nonendemic goiter. To our knowledge, this is the first time that a mutation in the thyroglobulin gene has been described in a patient with endemic simple goiter and further confirms the association between the exon 10 mutation and development of goiter.

Simple goitre is defined as an enlargement of the thyroid gland that is not the result of an inflammatory or neoplastic process and is not associated with thyrotoxicosis or myxoedema; the cause is unknown in most cases. Structural or regulatory defects in the proteins involved in thyroid metabolism might be involved in the functional abnormality that brings about the disorder. We have found a mutation within exon 10 of the thyroglobulin gene in 25 of 56 members of three families affected by simple goitre; 14 of the gene carriers had the disorder. DNA sequencing showed a mis-sense mutation within thyroglobulin gene exon 10, resulting in a glutamine to histidine substitution. Thus, some cases of non-endemic simple goitre are associated with a mutation at the thyroglobulin locus.