The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism.

Thyroglobulin (TG), the predominant glycoprotein of the thyroid gland, functions as matrix protein in thyroid hormonegenesis. TG deficiency results in thyroid dyshormonogenesis. These variants produce a heterogeneous spectrum of congenital goitre, with an autosomal recessive mode of inheritance. The purpose of this study was to identify and functionally characterize new variants in the TG gene in order to increase the understanding of the molecular mechanisms responsible for thyroid dyshormonogenesis. A total of four patients from two non-consanguineous families with marked alteration of TG synthesis were studied. The two families were previously analysed in our laboratory, only one deleterious allele, in each one, was detected after sequencing the TG gene (c.2359 C > T [p.Arg787*], c.5560 G > T [p.Glu1854*]). These findings were confirmed in the present studies by Next-Generation Sequencing. The single nucleotide coding variants of the TG gene were then analyzed to predict the possible variant causing the disease. The p.Pro2232Leu (c.6695 C > T), identified in both families, showing a low frequency population in gnomAD v2.1.1 database and protein homology, amino acid prediction, and 3D modeling analysis predict a potential pathogenic effect of this variant. We also transiently express p.Pro2232Leu in a full-length rat TG cDNA clone and confirmed that this point variant was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Consequently, each family carried a compound heterozygous for p.Arg787*/p.Pro2232Leu or p.Glu1854*/p.Pro2232Leu variants. In conclusion, our results confirm the pathophysiological importance of altered TG folding as a consequence of missense variants located in the ChEL domain of TG.

The p.Cys1281Tyr variant in the hinge module/flap region of thyroglobulin causes intracellular transport disorder and congenital hypothyroidism.

Congenital hypothyroidism (CH) due to thyroglobulin (TG) variants causes very low serum TG levels with normal or enlarged thyroid glands, depending on the severity of the defect, and with autosomal recessive inheritance. The purpose of this study was to functionally characterize p.Cys1281Tyr variant in the TG gene in order to increase our knowledge of the molecular mechanisms associated with CH. In order to find evidence that support the hypothesis that the p.Cys1281Tyr variant would affect the TG folding were performed amino acid prediction, 3D modeling and transient expression analysis in HEK293T cells. 18 of the 21″in silico" algorithms predict a deleterious effect of the p.Cys1281Tyr variant. The full-length 3D model p.Cys1281Tyr TG showed disulfide bond cleavage between the cysteines at positions 1249 and 1281 and rearrangement of the TG structure, while transient expression analysis indicated that p.Cys1281Tyr causes retention of the protein inside the cell. Consequently, these results show that this pathogenic variant makes it impossible for TG to fulfill its function in the biosynthesis process of thyroid hormones, causing CH. In conclusion, our results confirm the pathophysiological importance of misfolding of TG as a consequence of p.Cys1281Tyr variant located in the hinge module/flap region of TG.

Congenital goitrous hypothyroidism: mutation analysis in the thyroid peroxidase gene.

BACKGROUND: Iodide organification defect (IOD) is characterized by a reduced ability of the thyroid gland to retain iodide resulting in hypothyroidism. Mutations in thyroid peroxidase (TPO) gene appear to be the most common cause of IOD and are commonly inherited in an autosomal recessive fashion. The TPO gene is located on the chromosome 2p25. It comprises 17 exons, covers approximately 150 kb of genomic DNA and codes 933 amino acids. OBJECTIVES: In this study, we characterize the clinical and molecular basis of seven patients from four unrelated families with congenital hypothyroidism (CH) because of IOD. DESIGN AND METHODS: All patients underwent clinical, biochemical and imaging evaluation. The promoter and the complete coding regions of the human TPO along with the flanking intronic regions were analysed by single-strand conformation polymorphism analysis and direct DNA sequencing. Segregation analysis of mutations was carried out, and the effect of the novel missense identified mutations was investigated by 'in silico' studies. RESULTS: All subjects had congenital and persistent primary hypothyroidism. Three novel mutations: c.796C>T [p.Q266X], c.1784G>A [p.R595K] and c.2000G>A [p.G667D] and a previously reported mutation: c.1186_1187insGGCC [p.R396fsX472] have been identified. Four patients were compound heterozygous for p.R396fsX472/p.R595K mutations, two patients were homozygous for p.R595K, and the remaining patient was a compound heterozygous for p.Q266X/p.G667D. CONCLUSIONS: Our findings confirm the genetic heterogeneity of TPO defects and the importance of the implementation of molecular studies to determinate the aetiology of the CH with dyshormonogenesis.

Curating the gnomAD database: Report of novel variants in the thyroid peroxidase gene using in silico bioinformatics algorithms and a literature review.

Thyroid peroxidase (TPO) is a membrane-bound glycoprotein located at the apical side of the thyroid follicular cells that catalyzes both iodination and coupling of iodotyrosine residues within the thyroglobulin molecule, leading to the synthesis of thyroid hormone. Variants in TPO cause congenital hypothyroidism (CH) by iodide organification defect and are commonly inherited in an autosomal recessive fashion. In the present work, we report a detailed population analysis and bioinformatic prediction of the TPO variants indexed in the Genome Aggregation Database (gnomAD) v2.1.1. The proportion of missense cysteine variants and nonsense, frameshift, and splice acceptor/donor variants were analyzed in each ethnic group (European (Non-Finnish), European (Finnish), African/African Americans, Latino/Admixed American, East Asian, South Asian, Ashkenazi Jewish, Other). The results showed a clear predominance of frameshift variants in the East Asian (82%) and European (Finnish) (75%) population, whereas the splice site variants predominate in African/African Americans (99.46%), Other (96%), Latino/Admixed American (94%), South Asian (86%), European (Non-Finnish) (56%) and Ashkenazi Jewish (56%) populations. The analysis of the distribution of the variants indexed in gnomAD v2.1.1 database revealed that most missense variants identified in the An peroxidase domain map in exon 8, followed by exons 11, 7 and 9, and finally in descending order by exons 10, 6, 12 and 5. In total, 183 novel TPO variants were described (13 missense cysteine's variants, 158 missense variants involving the An peroxidase domain and 12 splicing acceptor or donor sites variants) which were not reported in the literature and that would have deleterious effects on prediction programs. In the gnomAD v2.1.1 population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:77. In conclusion, we provide an updated and curated reference source of new TPO variants for application in clinical diagnosis and genetic counseling. Also, this work contributes to elucidating the molecular basis of CH associated with TPO defects.

Mutational screening of the TPO and DUOX2 genes in Argentinian children with congenital hypothyroidism due to thyroid dyshormonogenesis.

PURPOSE: Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the preventable causes of both cognitive and motor deficits. We present a genetic and bioinformatics investigation of rational clinical design in 17 Argentine patients suspected of CH due to thyroid dyshormonogenesis (TDH). METHODS: Next-Generation Sequencing approach was used to identify variants in Thyroid Peroxidase (TPO) and Dual Oxidase 2 (DUOX2) genes. A custom panel targeting 7 genes associated with TDH [(TPO), Iodothyrosine Deiodinase I (IYD), Solute Carrier Family 26 Member 4 (SLC26A4), Thyroglobulin (TG), DUOX2, Dual Oxidase Maturation Factor 2 (DUOXA2), Solute Carrier Family 5 Member 5 (SLC5A5)] and 4 associated with thyroid dysembryogenesis [PAX8, FOXE1, NKX2-1, Thyroid Stimulating Hormone Receptor (TSHR)] has been designed. Additionally, bioinformatic analysis and structural modeling were carried out to predict the disease-causing potential variants. RESULTS: Four novel variants have been identified, two in TPO: c.2749-2 A > C and c.2752_2753delAG, [p.Ser918Cysfs*62] and two variants in DUOX2 gene: c.425 C > G [p.Pro142Arg] and c.2695delC [p.Gln899Serfs*21]. Eighteen identified TPO, DUOX2 and IYD variants were previously described. We identified potentially pahogenic biallelic variants in TPO and DUOX2 in 7 and 2 patients, respectively. We also detected a potentially pathogenic monoallelic variant in TPO and DUOX2 in 7 and 1 patients respectively. CONCLUSIONS: 22 variants have been identified associated with TDH. All described novel mutations occur in domains important for protein structure and function, predicting the TDH phenotype.

Structure and genetic variants of thyroglobulin: Pathophysiological implications.

Thyroglobulin (TG) plays a main role in the biosynthesis of thyroid hormones (TH), and, thus, it is involved in a wide range of vital functions throughout the life cycle of all vertebrates. Deficiency of TH production due to TG genetic variants causes congenital hypothyroidism (CH), with devastating consequences such as intellectual disability and impaired growth if untreated. To this day, 229 variations in the human TG gene have been identified while the 3D structure of TG has recently appeared. Although TG deficiency is thought to be of autosomal recessive inheritance, the introduction of massive sequencing platforms led to the identification of a variety of monoallelic TG variants (combined with mutations in other thyroid gene products) opening new questions regarding the possibility of oligogenic inheritance of the disease. In this review we discuss remarkable advances in the understanding of the TG architecture and the pathophysiology of CH associated with TG defects, providing new insights for the management of congenital disorders as well as counseling benefits for families with a history of TG abnormalities. Moreover, we summarize relevant aspects of TH synthesis within TG and offer an updated analysis of animal and cellular models of TG deficiency for pathophysiological studies of thyroid dyshormonogenesis while highlighting perspectives for new investigations. All in all, even though there has been sustained progress in understanding the role of TG in thyroid pathophysiology during the past 50 years, functional characterization of TG variants remains an important area of study for future advancement in the field.

Curating the gnomAD database: Report of novel variants in the thyrogobulin gene using in silico bioinformatics algorithms.

Thyroglobulin (TG) is a large glycosylated protein of 2767 amino acids, secreted by the thyrocytes into the follicular lumen. It plays an essential role in the process of thyroid hormone synthesis. TG gene variants lead to permanent congenital hypothyroidism. In the present work, we report a detailed population and bioinformatic prediction analyses of the TG variants indexed in the Genome Aggregation Database (gnomAD). The results showed a clear predominance of nonsense variants in the European (Finnish), European (Non-Finnish) and Ashkenazi Jewish ethnic groups, whereas the splice site variants predominate in South Asian and African/African-American populations. In total, 282 novel TG variants were described (47 missense involving the wild-type cysteine residues, 177 missense located in the ChEL domain and 58 splice site variants) which were not reported in the literature and that would have deleterious effects in prediction programs. In the gnomAD population, the estimated prevalence of heterozygous carriers of the potentially damaging variants was 1:320. In conclusion, we provide an updated and curated reference source for the diagnosis of thyroid disease, mainly to congenital hypothyroidism due to TG deficiency. The identification and characterization of TG variants is undoubtedly a valuable approach to study the TG structure/function relations and an important tool for clinical diagnosis and genetic counseling.

A novel mutation in intron 11 donor splice site, responsible of a rare genotype in thyroglobulin gene by altering the pre-mRNA splincing process. Cell expression and bioinformatic analysis.

Thyroglobulin (TG) is a homodimeric glycoprotein synthesized by the thyroid gland. To date, two hundred twenty-seven variations of the TG gene have been identified in humans. Thyroid dyshormonogenesis due to TG gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. The purpose of the present study was to identify and characterize new variants in the TG gene. We report an Argentine patient with congenital hypothyroidism, enlarged thyroid gland and low levels of serum TG. Sequencing of DNA, 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 maternal mutation consists of a c.3001+5G > A, whereas the paternal mutation consists of p.Arg296*. Minigen analysis of the variant c.3001+5A performed in HeLa, CV1 and Hek293T cell lines, showed a total lack of transcript expression. So, in order to validate that the loss of expression was caused by such variation, site-directed mutagenesis was performed on the mutated clone, which previously had a pSPL3 vector change, to give rise to a wild-type clone c.3001+5G, endorsing that the mutation c.3001+5G > A is the cause of the total lack of expression. In conclusion, we demonstrate that the c.3001+5G > A mutation causes a rare genotype, altering the splicing of the pre-mRNA. This work contributes to elucidating the molecular bases of TG defects associated with congenital hypothyroidism and expands our knowledge in relation to the pathologic roles of the position 5 in the donor splice site.

Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C>T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7.

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.

Defects in protein folding in congenital hypothyroidism.

Primary congenital hypothyroidism (CH) is the most common endocrine disease in children and one of the most common preventable causes of both cognitive and motor deficits. CH is a heterogeneous group of thyroid disorders in which inadequate production of thyroid hormone occurs due to defects in proteins involved in the gland organogenesis (dysembryogenesis) or in multiple steps of thyroid hormone biosynthesis (dyshormonogenesis). Dysembryogenesis is associated with genes responsible for the development or growth of thyroid cells: such as NKX2-1, FOXE1, PAX8, NKX2-5, TSHR, TBX1, CDCA8, HOXD3 and HOXB3 resulting in agenesis, hypoplasia or ectopia of thyroid gland. Nevertheless, the etiology of the dysembryogenesis remains unknown for most cases. In contrast, the majority of patients with dyshormonogenesis has been linked to mutations in the SLC5A5, SLC26A4, SLC26A7, TPO, DUOX1, DUOX2, DUOXA1, DUOXA2, IYD or TG genes, which usually originate goiter. About 800 genetic mutations have been reported to cause CH in patients so far, including missense, nonsense, in-frame deletion and splice-site variations. Many of these mutations are implicated in specific domains, cysteine residues or glycosylation sites, affecting the maturation of nascent proteins that go through the secretory pathway. Consequently, misfolded proteins are permanently entrapped in the endoplasmic reticulum (ER) and are translocated to the cytosol for proteasomal degradation by the ER-associated degradation (ERAD) machinery. Despite of all these remarkable advances in the field of the CH pathogenesis, several points on the development of this disease remain to be elucidated. The continuous study of thyroid gene mutations with the application of new technologies will be useful for the understanding of the intrinsic mechanisms related to CH. In this review we summarize the present status of knowledge on the disorders in the protein folding caused by thyroid genes mutations.

p.L571P in the linker domain of rat thyroglobulin causes intracellular retention.

Thyroglobulin (TG), a large glycosylated protein secreted by thyrocytes into the thyroid follicular lumen, plays an essential role in thyroid hormone biosynthesis. Rattus norvegicus TG (rTG) is encoded by a large single copy gene, 186-kb long, located on chromosome 7 composed of 48 exons encoding a 8461-kb mRNA. Although the TG gene displays sequence variability, many missense mutations do not impose any adverse effect on the TG protein, whereas other nucleotide substitutions may affect its TG stability and/or TG intracellular trafficking. In order to gain a further understanding of the protein domains regulating its intracellular fate, we cloned a full-length cDNA from rTG into the pcDNA6/V5-His B expression vector. However, transient expression of the cDNA in HEK293T cells showed that the encoded protein was not a wild-type molecule, as it was unable to be secreted in the culture supernatant. Sequencing analyses revealed three random mutations, which accidentally emerged during the course of cloning: c.1712T>C [p.L571P] in the linker domain (amino acid positions 360 to 604), c.2027A>G [p.Q676R] in TG type 1-6 repeat and c.2720A>G [p.Q907R] in the TG type 1-7 repeat. Expression of cDNAs encoding a combination of two mutations [p.Q676R-p.Q907R], [p.L571P-p.Q907R] or [p.L571P-p.Q676R] indicated that any TG bearing the p.L571P substitution was trapped intracellularly. Indeed, we expressed the single point mutant p.L571P and confirmed that this point mutation was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Endo H analysis showed that the p.L571P mutant is completely sensitive to the enzyme, whereas the will-type TG acquires full N-glycan modifications in Golgi apparatus. This data suggest that the p.L571P mutant contains the mannose-type N-glycan, that was added at the first stage of glycosylation. Complex-type N-glycan formation in the Golgi apparatus does not occur, consistent with defective endoplasmic reticulum exit of the mutant TG. Moreover, predictive analysis of the 3D linker domain showed that the p.L571P mutation would result in a significant protein conformational change. In conclusion, our studies identified a novel amino acid residue within the linker domain of TG associated with its conformational maturation and intracellular trafficking.

Genotyping of resistance to thyroid hormone in South American population. Identification of seven novel missense mutations in the human thyroid hormone receptor beta gene.

Thyroid Hormone Receptor beta (THRB) defects, typically transmitted as autosomal dominant traits, cause Resistance to Thyroid Hormone (RTH). We analyzed the THRB gene in thirteen South American patients with clinical evidence RTH from eleven unrelated families. Sequence analysis revealed seven novel missense mutations. Four novel mutations were identified in exon 9. The first, a c.991A>G transition which originates a substitution of asparagine by aspartic acid (p.N331D). The second nucleotide alteration consists of a guanine to cytosine transversion at position 1003 (c.1003G>C) and results in substitution of the alanine at codon 335 by proline (p.A335P). The third mutation, a c.1022T>C transition produces a change of leucine by proline (p.L341P). The fourth mutation detected in exon 9 was a c.1036C>T transition which replaces the leucine at codon 346 by phenylalanine (p.L346F). The sequencing of the exon 10 detected three novel missense mutations. The first, a c.1293A>G transition changing isoleucine 431 for methionine (p.I431M). The second, the cytosine at position 1339 was replaced by adenine (c.1339C>A) resulting in the replacement of proline by threonine (p.P447T). The third mutation detected in exon 10 was a c.1358C>T transition resulting in the substitution of proline at codon 453 by leucine (p.P453L). Finally, sequencing analysis of the THRB gene revealed three substitutions previously described (p.A268G, p.P453T and p.F459C). The p.P453T was found in two patients. In conclusion, we report thirteen patients with RTH caused by heterozygous mutations of the THRB gene. Seven of the identified mutations correspond to novel substitutions.

Identification and characterization of four PAX8 rare sequence variants (p.T225M, p.L233L, p.G336S and p.A439A) in patients with congenital hypothyroidism and dysgenetic thyroid glands.

CONTEXT: Thyroid dysgenesis may be associated with mutations in the paired box transcription factor 8 (PAX8) gene and is characterized by congenital hypothyroidism transmitted in an autosomal dominant mode. OBJECTIVES: The aim of this study was to identify new mutations in the PAX8 gene. Sixty congenital hypothyroidism-affected individuals with dysgenetic (agenesis, ectopia and hypoplasia) and eutopic thyroid glands were studied. METHODS: The 12 exons of the PAX8 gene along with their exon-intron boundaries were amplified from genomic DNA and a mutational screening was performed by single-strand conformational polymorphism (SSCP) followed by direct sequencing of samples with abnormal migration patterns. The PAX8 mutations were functionally characterized by transient transfection experiments. RESULTS: Molecular analysis of the PAX8 gene indicated that four affected individuals had four sequence differences: three novel variations [c.699C>T (p.L233L), c.1006G>A (p.G336S) and c.1317A>G (p.A439A)] and one recently reported [c.674C>T (p.T225M)], whereas the 56 remaining patients showed only wild-type alleles of PAX8. p.T225M, p.L233L and p.G336S variants were not detected in 530 chromosomes from 265 subjects randomly selected from the general population, whereas the p.A439A variant was identified in only one of the 530 chromosomes analysed. Functional analysis of the nonsynonymous substitutions showed that the p.T225M and p.G336S proteins had not lost their ability to bind a specific DNA sequence and to activate the transcription of the thyroglobulin (TG) promoter in synergy with thyroid transcription factor 1 (TTF1). CONCLUSIONS: We report the occurrence of two nonsynonymous substitutions, one recently reported (p.T225M) and one novel (p.G336S), and two novel synonymous substitutions (p.L233L and p.A439A) in the PAX8 gene. p.T225M and p.G336S are rare sequence variants or may act by inhibiting an unknown particular function. Our study also confirms the very low prevalence of PAX8 mutations in thyroid dysgenesis.

Molecular analysis of thyroglobulin mutations found in patients with goiter and hypothyroidism.

Thyroid dyshormonogenesis due to thyroglobulin (TG) gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. Up to now, one hundred seventeen deleterious mutations in the TG gene have been identified and characterized. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report eight patients from seven unrelated families with goiter, hypothyroidism and low levels of serum TG. All patients underwent clinical, biochemical and image evaluation. Sequencing of DNA, genotyping, as well as bioinformatics analysis were performed. Molecular analyses revealed three novel inactivating TG mutations: c.5560G>T [p.E1835*], c.7084G>C [p.A2343P] and c.7093T>C [p.W2346R], and four previously reported mutations: c.378C>A [p.Y107*], c.886C>T [p.R277*], c.1351C>T [p.R432*] and c.7007G>A [p.R2317Q]. Two patients carried homozygous mutations (p.R277*/p.R277*, p.W2346R/p.W2346R), four were compound heterozygous mutations (p.Y107*/p.R277* (two unrelated patients), p.R432*/p.A2343P, p.Y107*/p.R2317Q) and two siblings from another family had a single p.E1835* mutated allele. Additionally, we include the analysis of 48 patients from 31 unrelated families with TG mutations identified in our present and previous studies. Our observation shows that mutations in both TG alleles were found in 27 families (9 as homozygote and 18 as heterozygote compound), whereas in the remaining four families only one mutated allele was detected. The majority of the detected mutations occur in exons 4, 7, 38 and 40. 28 different mutations were identified, 33 of the 96 TG alleles encoded the change p.R277*. In conclusion, our results confirm the genetic heterogeneity of TG defects and the pathophysiological importance of the predicted TG misfolding and therefore thyroid hormone formation as a consequence of truncated TG proteins and/or missense mutations located within its ACHE-like domain.

Recurrence of the p.R277X/p.R1511X compound heterozygous mutation in the thyroglobulin gene in unrelated families with congenital goiter and hypothyroidism: haplotype analysis using intragenic thyroglobulin polymorphisms.

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.

Iodide handling disorders (NIS, TPO, TG, IYD).

Iodide Handling Disorders lead to defects of the biosynthesis of thyroid hormones (thyroid dyshormonogenesis, TD) and thereafter congenital hypothyroidism (CH), the most common endocrine disease characterized by low levels of circulating thyroid hormones. The prevalence of CH is 1 in 2000-3000 live births. Prevention of CH is based on prenatal diagnosis, carrier identification, and genetic counseling. In neonates a complete diagnosis of TD should include clinical examination, biochemical thyroid tests, thyroid ultrasound, radioiodine or technetium scintigraphy and perchlorate discharge test (PDT). Biosynthesis of thyroid hormones requires the presence of iodide, thyroid peroxidase (TPO), a supply of hydrogen peroxide (DUOX system), an iodine acceptor protein, thyroglobulin (TG), and the rescue and recycling of iodide by the action of iodotyrosine deiodinase or iodotyrosine dehalogenase 1 (IYD or DEHAL1). The iodide transport is a two-step process involving transporters located either in the basolateral or apical membranes, sodium iodide symporter (NIS) and pendrin (PDS), respectively. TD has been linked to mutations in the solute carrier family 5, member 5 transporter (SLC5A5, encoding NIS), solute carrier family 26, member 4 transporter (SLC26A4, encoding PDS), TPO, DUOX2, DUOXA2, TG and IYD genes. These mutations produce a heterogeneous spectrum of CH, with an autosomal recessive inheritance. Thereafter, the patients are usually homozygous or compound heterozygous for the gene mutations and the parents, carriers of one mutation. In the last two decades, considerable progress has been made in identifying the genetic and molecular causes of TD. Recent advances in DNA sequencing technology allow the massive screening and facilitate the studies of phenotype variability. In this article we included the most recent data related to disorders caused by mutations in NIS, TPO, TG and IYD.

[Transient congenital hypothyroidism due to biallelic defects of DUOX2 gene. Two clinical cases]. / Hipotiroidismo congénito transitorio por defectos bialélicos del gen DUOX2. Dos casos clínicos.

Congenital hypothyroidism affects 1:2000-3000 newborns detected by neonatal screening programs. Dual oxidases, DUOX1 and 2, generate hydrogen peroxide needed for the thyroid hormone synthesis. Hipotiroidismo congénito transitorio por defectos bialélicos del gen DUOX2. Dos casos clínicos Transient congenital hypothyroidism due to biallelic defects of DUOX2 gene. Two clinical cases Mutations in the DUOX2 gene have been described in transient and permanent congenital hypothyroidism. Two brothers with congenital hypothyroidism detected by neonatal screening with eutopic gland and elevated thyroglobulin are described. They were treated with levothyroxine until it could be suspended in both during childhood, assuming the picture as transient. Organification disorder was confirmed. Both patients were compounds heterozygous for a mutation in exon 9 of the paternal allele (c.1057_1058delTT, p.F353PfsX36 or p.F353fsX388) and in exon 11 of the maternal allele (c.1271T > G, p.Y425X) of DUOX2 gene. Our finding confirms that the magnitude of the defect of DUOX2 is not related to the number of inactivated alleles, suggesting compensatory mechanisms in the peroxide supply.

El hipotiroidismo congénito afecta a 1:2000-3000 recién nacidos detectados por pesquisa neonatal. Las oxidasas duales, DUOX1 y 2, generan agua oxigenada, lo que constituye un paso crítico en la síntesis hormonal. Se han comunicado mutaciones en el gen DUOX2 en casos de hipotiroidismo congénito transitorio y permanente. Se describen dos hermanos con hipotiroidismo congénito detectados por pesquisa neonatal, con glándula tiroides eutópica y tiroglobulina elevada. Recibieron levotiroxina hasta su reevaluación en la infancia con suspensión del tratamiento. Su función tiroidea fue normal y se consideró el cuadro como transitorio por un posible defecto de organificación. Ambos pacientes eran heterocigotos compuestos para una mutación en el exón 9 del alelo paterno (c.1057_1058delTT, p.F353PfsX36 o p.F353fsX388) y otra en el exón 11 del alelo materno (c.1271T>G, p.Y425X) del gen DUOX2. Nuestro hallazgo confirma que la magnitud del defecto de DUOX2 no se relaciona con el número de alelos afectados, lo que sugiere mecanismos compensadores en la generación de peróxido.

Molecular advances in thyroglobulin disorders.

Synthesis of tri-iodothyronine (T(3)) and thyroxine (T(4)) follows a metabolic pathway that depends on the integrity of the thyroglobulin structure. This large glycoprotein is a homodimer of 660 kDa synthesized and secreted by the thyroid cells into the lumen of thyroid follicle. In humans it is coded by a single copy gene, 270 kb long, that maps on chromosome 8q24 and contains an 8.5 kb coding sequence divided into 48 exons. The preprotein monomer is composed of a 19-amino acid signal peptide followed by a 2749-amino acid polypeptide. In the last decade, several mutations in the thyroglobulin gene were reported. In animals, four of them have been observed in Afrikander cattle (p.R697X), Dutch goats (p.Y296X), cog/cog mouse (p.L2263P) and rdw rats (p.G2300R). Mutations in the human thyroglobulin gene are associated with congenital goiter or endemic and nonendemic simple goiter. Thirty-five inactivating mutations have been identified and characterized in the human thyroglobulin gene: 20 missense mutations (p.C175G, p.Q310P, p.Q851H, p.S971I, p.R989C, p.P993L, p.C1058R, p.C1245R, p.S1447N, p.C1588F, p.C1878Y, p.I1912V, p.C1977S, p.C1987Y, p.C2135Y, p.R2223H, p.G2300D, p.R2317Q, p.G2355V, p.G2356R), 8 splice site mutations (g.IVS3-3C>G, g.IVS5+1G>A, g.IVS10-1G>A, g.IVS24+1G>C, g.IVS30+1G>T, g.IVS30+1G>A, g.IVS34-1G>C, g.IVS45+2T>A) 5 nonsense mutations (p.R277X, p.Q692X, p.W1418X, p.R1511X, p.Q2638X) and 2 single nucleotide deletions (p.G362fsX382, p.D1494fsX1547). The thyroglobulin gene has been also identified as the major susceptibility gene for familial autoimmune thyroid diseases (AITD) by linkage analysis using highly informative polymorphic markers. In conclusion the identification of mutations in the thyrogobulin gene has provided important insights into structure-function relationships.

Compound heterozygous DUOX2 gene mutations (c.2335-1G>C/c.3264_3267delCAGC) associated with congenital hypothyroidism. Characterization of complex cryptic splice sites by minigene analysis.

Iodide Organification defects (IOD) represent 10% of cases of congenital hypothyroidism (CH) being the main genes affected that of TPO (thyroid peroxidase) and DUOX2 (dual oxidasa 2). From a patient with clinical and biochemical criteria suggestive with CH associated with IOD, TPO and DUOX2 genes were analyzed by means of PCR-Single Strand Conformation Polymorphism analysis and sequencing. A novel heterozygous compound to the mutations c.2335-1G>C (paternal mutation, intron 17) and c.3264_3267delCAGC (maternal mutation, exon 24) was identified in the DUOX2 gene. Ex-vivo splicing assays and subsequent RT-PCR and sequencing analyses were performed on mRNA isolated from the HeLa cells transfected with wild-type and mutant pSPL3 expression vectors. The wild-type and c.2335-1G>C mutant alleles result in the complete inclusion or exclusion of exon 18, or in the activation of an exonic cryptic 5' ss with the consequent deletion of 169 bp at the end of this exon. However, we observed only a band of the expected size in normal thyroid tissue by RT-PCR. Additionally, the c.2335-1G>C mutation activates an unusual cryptic donor splice site in intron 17, located at position -14 of the authentic intron 17/exon 18 junction site, with an insertion of the last 14 nucleotides of the intron 17 in mutant transcripts with complete and partial inclusion of exon 18. The theoretical consequences of splice site mutation, predicted with the bioinformatics NNSplice, Fsplice, SPL, SPLM and MaxEntScan programs were investigated and evaluated in relation with the experimental evidence. These analyses confirm that c.2335-1G>C mutant allele would result in the abolition of the authentic splice acceptor site. The results suggest the coexistence in our patient of four putative truncated proteins of 786, 805, 806 and 1105 amino acids, with conservation of peroxidase-like domain and loss of gp91(phox)/NOX2-like domain. In conclusion a novel heterozygous compound was identified being responsible of IOD. Cryptic splicing sites have been characterized in DUOX2 gene for the first time. The use of molecular biology techniques is a valuable tool for understanding the molecular pathophysiology of this type of thyroid defects.