RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito , Bocio , Humanos , Hipotiroidismo Congénito/genética , Tiroglobulina/genética , Tiroglobulina/metabolismo , Células HEK293 , Bocio/genética , Hormonas Tiroideas

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito , Bocio , Animales , Humanos , Ratas , Hipotiroidismo Congénito/genética , Células HEK293 , Tiroglobulina/genética , Tiroglobulina/metabolismo , Transporte de Proteínas/genética

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito , Tiroglobulina , Humanos , Tiroglobulina/genética , Yoduro Peroxidasa/genética , Monoyodotirosina/genética , Yoduros , Biología Computacional , Cisteína , Hipotiroidismo Congénito/genética , Hormonas Tiroideas , Mutación/genética , Peroxidasas/genética , Algoritmos

RESUMEN

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.

Asunto(s)

Autoantígenos , Hipotiroidismo Congénito , Oxidasas Duales , Yoduro Peroxidasa , Proteínas de Unión a Hierro , Argentina , Autoantígenos/genética , Niño , Hipotiroidismo Congénito/genética , Oxidasas Duales/genética , Humanos , Yoduro Peroxidasa/genética , Proteínas de Unión a Hierro/genética , Mutación , Receptores de Tirotropina/genética

RESUMEN

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.

Asunto(s)

Biología Computacional/métodos , Hipotiroidismo Congénito/genética , Etnicidad/genética , Variación Genética , Tiroglobulina/genética , Algoritmos , Empalme Alternativo , Codón sin Sentido , Curaduría de Datos , Bases de Datos Genéticas , Humanos , Mutación Missense , Dominios Proteicos , Tiroglobulina/química

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito/genética , Variación Genética , Tiroglobulina/química , Tiroglobulina/genética , Animales , Humanos , Modelos Moleculares , Conformación Proteica , Tiroglobulina/metabolismo , Hormonas Tiroideas/metabolismo

RESUMEN

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.

Asunto(s)

Biología Computacional , Intrones/genética , Mutación/genética , Precursores del ARN/genética , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Tiroglobulina/genética , Secuencia de Bases , Genotipo , Células HEK293 , Células HeLa , Humanos , Recién Nacido , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Precursores del ARN/metabolismo , Tiroglobulina/química

RESUMEN

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.

Asunto(s)

Espacio Intracelular/metabolismo , Mutación/genética , Tiroglobulina/química , Tiroglobulina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Glicósido Hidrolasas/metabolismo , Células HEK293 , Humanos , Masculino , Mutagénesis/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Ratas Wistar

RESUMEN

Massive parallel sequencing technologies are facilitating the faster identification of sequence variants with the consequent capability of untangling the molecular bases of many human genetic syndromes. However, it is not always easy to understand the impact of novel variants, especially for missense changes, which can lead to a spectrum of phenotypes. This study presents a custom-designed multistep methodology to evaluate the impact of novel variants aggregated in the genome aggregation database for the HBB, HBA2, and HBA1 genes, by testing and improving its performance with a dataset of previously described alterations affecting those same genes. This approach scored high sensitivity and specificity values and showed an overall better performance than sequence-derived predictors, highlighting the importance of protein conformation and interaction specific analyses in curating variant databases. This study also describes the strengths and limitations of these structural studies and allows identifying residues in the globin chains more prone to tolerate substitutions.

Asunto(s)

Biología Computacional , Bases de Datos Genéticas , Variación Genética , Hemoglobinas/genética , Alelos , Sustitución de Aminoácidos , Biología Computacional/métodos , Biología Computacional/normas , Genotipo , Hemoglobinas/química , Humanos , Mutación con Pérdida de Función , Mutación , Sistemas de Lectura Abierta , Fenotipo , Sensibilidad y Especificidad , Globinas alfa/química , Globinas alfa/genética , Globinas beta/química , Globinas beta/genética

RESUMEN

In humans, the thyroid hormones T3 and T4 are synthesized in the thyroid gland in a process that crucially involves the iodoglycoprotein thyroglobulin. The overall structure of thyroglobulin is conserved in all vertebrates. Upon thyroglobulin delivery from thyrocytes to the follicular lumen of the thyroid gland via the secretory pathway, multiple tyrosine residues can become iodinated to form mono-iodotyrosine (MIT) and/or di-iodotyrosine (DIT); however, selective tyrosine residues lead to preferential formation of T4 and T3 at distinct sites. T4 formation involves oxidative coupling between two DIT side chains, and de novo T3 formation involves coupling between an MIT donor and a DIT acceptor. Thyroid hormone synthesis is stimulated by TSH activating its receptor (TSHR), which upregulates the activity of many thyroid gene products involved in hormonogenesis. Additionally, TSH regulates post-translational changes in thyroglobulin that selectively enhance its capacity for T3 formation - this process is important in iodide deficiency and in Graves disease. 167 different mutations, many of which are newly discovered, are now known to exist in TG (encoding human thyroglobulin) that can lead to defective thyroid hormone synthesis, resulting in congenital hypothyroidism.

Asunto(s)

Tiroglobulina/fisiología , Glándula Tiroides/metabolismo , Tiroxina/biosíntesis , Triyodotironina/biosíntesis , Animales , Enfermedad de Graves/diagnóstico , Enfermedad de Graves/genética , Enfermedad de Graves/metabolismo , Humanos , Glándula Tiroides/patología , Hormonas Tiroideas/biosíntesis , Hormonas Tiroideas/genética , Tiroxina/genética , Triyodotironina/genética

RESUMEN

Hemoglobin (Hb) synthesis is a complex, well-coordinated process that requires molecular chaperones. These intervene in different steps: regulating epigenetic mechanisms necessary for the adequate expression of the α- and ß-globin clusters, binding the nascent peptides and helping them acquire their native structure, preventing oxidative damage by free globin chains and preventing the cleavage of essential erythroid transcription factors. This study analyzed the distribution of the single nucleotide polymorphism (SNP) rs4296276 in intron 1 of the α-globin chaperone α Hb-stabilizing protein (AHSP) in the Argentinean population. The risk allele was found in thalassemia patients who exhibited more severe phenotypes than expected. Future studies may help establish the role of these chaperones as modifiers in pathological states with globin chain imbalance, such as thalassemia.

Asunto(s)

Proteínas Sanguíneas/genética , Hemoglobinas/biosíntesis , Chaperonas Moleculares/genética , Polimorfismo de Nucleótido Simple , Alelos , Argentina/epidemiología , Humanos , Intrones/genética , Epidemiología Molecular , Talasemia/genética , Globinas alfa/genética

RESUMEN

Hemoglobinopathies are the most common autosomal recessive disorders and are mostly inherited in a recessive manner. However, certain mutations can affect the globin chain stability, leading to dominant forms of thalassemia. The aim of this work was the molecular and structural characterization of two heterozygous in-frame deletions, leading to ß-globin variants in pediatric patients in Argentina. The HBB gene of the probands and their parents was sequenced, and other markers of globin chain imbalance were analyzed. Several structural analyses were performed, and the effect of the mutations on the globin chain stability was analyzed. In Hb JC-Paz, HBB:c.29_37delCTGCCGTTA (p.Ala10_Thr12del), detected in an Argentinean boy, one α-helix turn is expected to be lost. In Hb Tavapy, HBB:c.182_187delTGAAGG (p.Val60_Lys61del), the deleted residues are close to distal histidine (His63) in the heme pocket. Both mutations are predicted to have a destabilizing effect. The development of computational structural models and bioinformatics algorithms is expected to become a useful tool to understand the impact of the mutations leading to dominant thalassemia.

Asunto(s)

Sustitución de Aminoácidos , Hemoglobinas Anormales/genética , Sistemas de Lectura , Eliminación de Secuencia , Globinas beta/genética , Niño , Preescolar , Análisis Mutacional de ADN , Índices de Eritrocitos , Femenino , Hemoglobinopatías/sangre , Hemoglobinopatías/diagnóstico , Hemoglobinopatías/genética , Hemoglobinopatías/terapia , Hemoglobinas Anormales/química , Humanos , Masculino , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Globinas beta/química

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito/genética , Bocio/genética , Mutación/genética , Tiroglobulina/genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Segregación Cromosómica/genética , Hipotiroidismo Congénito/diagnóstico por imagen , Análisis Mutacional de ADN , Familia , Femenino , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad , Bocio/diagnóstico por imagen , Haplotipos/genética , Humanos , Recién Nacido , Masculino , Linaje , Tiroglobulina/química

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito/genética , Mutación , NADPH Oxidasas/genética , Sitios de Empalme de ARN , Niño , Hipotiroidismo Congénito/metabolismo , Oxidasas Duales , Células HeLa , Heterocigoto , Humanos , Masculino , NADPH Oxidasas/metabolismo , Linaje , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN , Glándula Tiroides/metabolismo

RESUMEN

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.

Asunto(s)

Pueblo Asiatico/genética , Hipotiroidismo Congénito/genética , Bocio/congénito , Bocio/genética , Polimorfismo de Nucleótido Simple , Sitios de Empalme de ARN , Tiroglobulina/genética , Adolescente , Animales , Células COS , Chlorocebus aethiops , Hipotiroidismo Congénito/patología , Exones , Femenino , Bocio/patología , Células HeLa , Heterocigoto , Humanos , Masculino , Linaje , Análisis de Secuencia de ADN , Tiroglobulina/sangre , Vietnam

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito/genética , Tiroglobulina/genética , Secuencia de Bases , Consanguinidad , Análisis Mutacional de ADN , Estudios de Asociación Genética , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo de Nucleótido Simple , Eliminación de Secuencia , Inversión de Secuencia , Tiroglobulina/deficiencia

RESUMEN

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.

Asunto(s)

Bocio/genética , Hipotiroidismo/genética , Mutación Missense , Tiroglobulina/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Secuencia Conservada , Análisis Mutacional de ADN , Femenino , Estudios de Asociación Genética , Heterogeneidad Genética , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Fenotipo , Polimorfismo Conformacional Retorcido-Simple , Estructura Secundaria de Proteína , Homología Estructural de Proteína

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito/genética , Yoduro Peroxidasa/genética , Adolescente , Niño , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Mutación

RESUMEN

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.

Asunto(s)

Hipotiroidismo Congénito/genética , Exones , Sitios de Empalme de ARN , Tiroglobulina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Hipotiroidismo Congénito/diagnóstico por imagen , Femenino , Humanos , Mutación INDEL , Recién Nacido , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN , Glándula Tiroides/diagnóstico por imagen , Ultrasonografía

RESUMEN

Human thyroglobulin (TG) gene is a single copy gene, 270 kb long, that maps on chromosome 8q24.2-8q24.3 and contains an 8.5-kb coding sequence divided into 48 exons. TG is exclusively synthesized in the thyroid gland and represents a highly specialized homodimeric glycoprotein for thyroid hormone biosynthesis. Mutations in the TG gene lead to permanent congenital hypothyroidism. The presence of low TG level and also normal perchlorate discharge test in a goitrous individual suggest a TG gene defect. Until now, 52 mutations have been identified and characterized in the human TG gene with functional impact such as structural changes in the protein that alter the normal protein folding, assembly and biosynthesis of thyroid hormones. 11 of the mutations affect splicing sites, 11 produce premature stop codons, 23 lead to amino acid changes, 6 deletions (5 single and 1 involving a large number of nucleotides) and 1 single nucleotide insertion. TG mutations are inherited in an autosomal recessive manner and affected individuals are either homozygous or compound heterozygous. The p.R277X, p.C1058R, p.C1977S, p.R1511X, p.A2215D and p.R2223H mutations are the most frequently identified TG mutations. This mini-review focuses on genetic and clinical aspects of TG gene defects.

Asunto(s)

Hipotiroidismo Congénito/genética , Mutación , Tiroglobulina/genética , Algoritmos , Animales , Secuencia de Bases , Humanos , Modelos Biológicos , Mutación/fisiología , Proteínas/genética , Proteínas/metabolismo , Proteínas/fisiología , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tiroglobulina/fisiología