Biomedical subjects

Carina M Rivolta

Publications and source records attributed to Carina M Rivolta.

12 recordsLinked to original sources

PubMed2006

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.

Amino Acid Sequence↗

PubMed2005

Three mutations (p.Q36H, p.G418fsX482, and g.IVS19-2A>C) in the dual oxidase 2 gene responsible for congenital goiter and iodide organification defect.

BACKGROUND: Iodide organification defects are associated with mutations in the dual oxidase 2 (DUOX2) gene and are characterized by a positive perchlorate discharge test. These mutations produce a congenital goitrous hypothyroidism, usually transmitted in an autosomal recessive mode. METHODS: We studied the complete coding sequence of the human DUOX2 gene by single-strand conformational polymorphism (SSCP) analysis of DNA from 17 unrelated patients with iodide organification defects. Samples showing an aberrant pattern were directly sequenced. All mutations were validated by SSCP analysis. Finally, the effect of a splicing mutation was studied by construction of minigenes. RESULTS: Genomic DNA sequencing revealed 3 novel mutations [c.108G>C (p.Q36H), c.1253delG (p.G418fsX482), and g.IVS19-2A>C] and 1 previously reported mutation [c.2895-2898delGTTC (p.S965fsX994)] in 2 families with 1 (family 1) and 2 (family 2) affected members. This implies the inheritance of 2 compound heterozygous mutations, p.Q36H and p.S965fsX994 in family 1 and p.G418fsX482 and g.IVS19-2A>C in family 2. The c.1253delG mutation was associated with a c.1254C>A transversion. In vitro transcription analysis showed that exon 20 is skipped entirely when the g.IVS19-2A>C mutation is present. The wild-type glutamine residue at position 36 is strictly conserved. CONCLUSIONS: Two previously unknown compound heterozygous mutations in the DUOX2 gene, p.Q36H/p.S965fsX994 and p.G418fsX482/g.IVS19-2A>C, are responsible for iodide organification defects in 2 unrelated families. Identification of the molecular basis of this disorder might be helpful for understanding the pathophysiology of this congenital hypothyroidism.

Animals↗

PubMed2005

A new case of congenital goiter with hypothyroidism caused by a homozygous p.R277X mutation in the exon 7 of the thyroglobulin gene: a mutational hot spot could explain the recurrence of this mutation.

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.

Amino Acid Sequence↗

PubMed2005

Nonsense-associated alternative splicing of the human thyroglobulin gene.

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.

Alternative Splicing↗

PubMed2005

Naturally occurring mutations in the thyroglobulin gene.

Thyroglobulin (Tg) is a large glycoprotein dimer secreted into the follicular lumen. It serves as the matrix for the synthesis of thyroxine (T4) and triiodothyronine (T3), and the storage of thyroid hormone and iodide. In response to demand for thyroid hormone secretion, Tg is internalized into the follicular cell and digested in lysosomes. Subsequently, the thyronines T4 (approximately 80%) and T3 (approximately 20%) are released into the blood stream. Biallelic mutations in the Tg gene have been identified in several animal species and human patients presenting with goiter and overt or compensated hypothyroidism. In untreated patients, goiters are often remarkably large and display continuous growth. In most instances, the affected individuals have related parents and are homozygous for inactivating mutations in the Tg gene. More rarely, compound heterozygous mutations lead to a loss of function of both alleles. Molecular analyses indicate that at least some of these alterations result in a secretory defect and an endoplasmic reticulum storage disease (ERSD). This review discusses the nature and consequences of naturally occurring Tg gene mutations in humans and several animal species. Recent recommendations for the nomenclature of mutations have led to different numbering systems, an aspect that is discussed in order to clarify discrepancies between different publications.

Animals↗

PubMed2005

[The thyroid as a model for molecular mechanisms in genetic diseases].

Thyroid diseases constitute a heterogeneous collection of abnormalities associated with mutations in genes responsible for the development of thyroid: thyroid transcription factor-1 (TTF-1), thyroid transcriptions factor-2 (TTF-2) and PAX8, or in one of the genes coding for the proteins involved in thyroid hormone biosynthesis such as thyroglobulin (TG), thyroperoxidase (TPO), hydrogen peroxide-generating system (DUOX2), sodium/iodide symporter (NIS), pendrin (PDS), TSH and TSH receptor (TSHr). Congenital hypothyroidism occurs with a prevalence of 1 in 4000 newborns. Patients with this syndrome can be divided into two groups: nongoitrous (dysem/bryogenesis) or goitrous (dyshormonogenesis) congenital hypothyroidism. The dysembryogenesis group, which accounts for 85% of the cases, results from ectopy, agenesis and hypoplasia. In a minority of these patients, the congenital hypothyroidism is associated with mutations in TTF-1, TTF-2, PAX-8, TSH or TSHr genes. The presence of congenital goiter (15% of the cases) has been linked to mutations in the NIS, TG, TPO, DUOX2 or PDS genes. The congenital hypothyroidism with dyshormonogenesis is transmitted as an autosomal recessive trait. Somatic mutations of the TSHr have been identified in hyperfunctioning thyroid adenomas. Another established thyroid disease is the resistance to thyroid hormone (RTH). It is a syndrome of reduced tissue responsiveness to hormonal action caused by mutations located in the thyroid hormone receptor beta (TRbeta) gene. Mutant TRbetas interfere with the function of the wild-type receptor by a dominant negative mechanism. In conclusion, the identification of mutations in the thyroid expression genes has provided important insights into structure-function relationships. The thyroid constitutes an excellent model for the molecular study of genetic diseases.

Congenital Hypothyroidism↗

PubMed2004

Two distinct compound heterozygous constellations (R277X/IVS34-1G>C and R277X/R1511X) in the thyroglobulin (TG) gene in affected individuals of a Brazilian kindred with congenital goiter and defective TG synthesis.

In this study, we have extended our initial molecular studies of a nonconsanguineous family with two affected siblings and one of their nephews with congenital goiter, hypothyroidism, and marked impairment of thyroglobulin synthesis. Genomic DNA sequencing revealed that the index patient (affected nephew) was heterozygous for a single base change of a cytosine to a thymine at nucleotide 886 in exon 7 (886C>T, mother's mutation) in one allele and for a novel guanine to cytosine transversion at position -1 of the splice acceptor site in intron 34 (IVS34-1G>C, father's mutation) in the other allele. The two affected siblings inherited the 886C>T mutation from their mother and a previously reported cytosine to thymine transition at nucleotide 4588 in exon 22 from their father (4588C>T). The 886C>T and 4588C>T substitutions resulted in premature stop codons at amino acids 277 (R277X) and 1511 (R1511X), respectively. In vitro transcription analysis showed that the exon 35 is skipped entirely when the IVS34-1G>C mutation is present, whereas the wild-type allele is correctly spliced. SSCP (exon 7 and 35) and restriction analysis (exon 22) using Taq I indicated that the two affected siblings, the affected nephew, his mother, and his unaffected brother were all heterozygous for the R277X mutation. The two affected siblings, their father, and three unaffected siblings were all heterozygous for the R1511X mutation, whereas the affected nephew and his father were heterozygous for the IVS34-1G>C mutation. Moreover, in this kindred, we have characterized polymorphisms (insertion/deletion, microsatellite, and single nucleotide polymorphism) located within introns 18 and 29 and exon 44 that are associated with the described mutations. Haplotype analysis with these polymorphic markers in two unrelated Brazilian families (present family studied and previously reported family) harboring the R277X mutation suggests a founder effect for the R277X mutation. In conclusion, the affected individuals of this family are either compound heterozygous for R277X/IVS34-1G>C or R277X/R1511X. This observation further supports that thyroglobulin gene mutations display significant intraallelic heterogeneity.

Amino Acid Sequence↗

PubMed2004

A novel 1297-1304delGCCTGCCA mutation in the exon 10 of the thyroid hormone receptor beta gene causes resistance to thyroid hormone.

INTRODUCTION: Resistance to the thyroid hormone (RTH) is an inherited syndrome of reduced tissue responsiveness to hormonal action caused by mutations located in the ligand-binding domain and adjacent hinge region of the thyroid hormone receptor beta (TRbeta) gene. PATIENT: The patient in this study, a 42-year-old Caucasian male, came to medical attention because he experienced atrial fibrillation. Clinical evaluation showed a small and diffuse goiter and biochemical tests revealed markedly elevated concentrations of total T4, total T3, and free T4, normal thyroid-stimulating hormone (TSH) values and slightly increased I131 thyroid uptake at 24 hours. The thyroperoxidase, thyroglobulin, and TSH receptor antibodies were positive. He was treated with cabergoline plus methimazole. This treatment was stopped because of the inconsistent response, monotherapy with tri-iodothyroacetic acid (TRIAC) was then prescribed after molecular diagnosis confirmed RTH syndrome. METHODS: The exons 9 and 10 of the TRbeta gene, including splicing signals and the flanking intronic regions of each intron, were amplified with PCR. DNA sequences from each amplified fragment were performed with the Taq polymerase-based chain terminator method and using the specific TRbeta forward and reverse primers. RESULTS: Direct sequence analysis of the exons 9 and 10 of the TRbeta gene revealed an eight basepair deletion, 1297-1304delGCCTGCCA in exon 10. The mutation produces a frameshift at amino acid 433 and introduces a stop codon TGA at position 461, 85 nucleotides downstream from deletion. This alteration was not detected in either the father or mother of the patient, suggesting a de novo mutation that was confirmed by DNA fingerprint analysis. CONCLUSIONS: In the present study we have identified a novel sporadic mutation corresponding to 1297-1304delGCCTGCCA deletion in the activating function 2 (AF-2) region of TRbeta. To our knowledge, this is the first time that the presence of a partial deletion of eight nucleotides in the TRbeta has been reported.

Adult↗

PubMed2003

Five novel inactivating mutations in the thyroid peroxidase gene responsible for congenital goiter and iodide organification defect.

Thyroid peroxidase (TPO) defects, typically transmitted as autosomal recessive traits, result in hypothyroid goiters with failure to convert iodide into organic iodine. We analyzed the TPO gene in 14 unrelated patients with clinical evidence of iodide organification defects. Seven of the affected individuals harbored mutations in the TPO gene; one was compound heterozygous, the others were simply heterozygous for TPO mutations. Five novel mutations have been identified, one of which was found to be a single nucleotide deletion, while the other four were single nucleotide substitutions. A frameshift mutation c.387delC was detected in exon 5 which leads to an early termination signal in exon 7 (p.N129fsX208). Two missense mutations were identified in exon 8. The first, a c.920A>C transversion that results in a p.N307T substitution, was found in two patients. The second, a c.1297G>A transition, results in p.V433M. A c.1496C>T transition was detected in exon 9 that caused the substitution p.P499L. Finally, in exon 14 a c.2422T>C transition was identified, causing a p.C808R change. In addition, the previously reported GGCC duplication in exon 8 (c.1186_1187insGGCC; p.R396fsX472) was also detected in two affected individuals, one of whom was a compound heterozygous (p.R396fsX472/p.V433M).

Frameshift Mutation↗

PubMed2003

Identification and characterization of a novel large insertion/deletion polymorphism of 1464 base pair in the human thyroglobulin gene.

We identified a novel large insertion/deletion (Indel) polymorphism of 1464 bp localized in intron 18 of the human thyroglobulin gene. Data from sequence showed a high A+T content (62%), two 17-bp long motif repeats, and three different types of 10-bp long palindromic sequences. The comparison between these 1464 bp and sequences deposited in National Center for Biotechnology Information (NCBI)/GenBank database exhibit a nonsignificant degree of homology with any previously described sequences. The long polymerase chain reaction (PCR) method was used to amplify the genomic DNA region containing intron 17/exon 18/intron 18/exon 19/intron 19 by primers situated in the introns 17 and 19. The amplification generates two fragments of 3.5 and 5.0 kb that correspond to the exclusion or inclusion of a 1464-bp segment, respectively. Both variants are thus widely represented in the human population; giving allele frequencies of 0.56 (insertion) and 0.44 (deletion). Finally, the polymorphism was confirmed by sequence analysis of the 5.0- and 3.5-kb amplified fragments.

Alleles↗

PubMed2003

Compound heterozygous mutations in the thyroglobulin gene (1143delC and 6725G-->A [R2223H]) resulting in fetal goitrous hypothyroidism.

In a 22-yr-old healthy woman, a fetal goiter was diagnosed coincidentally by ultrasound during the sixth month of gestation, and hypothyroidism was affirmed by a high TSH (336 mU/liter) concentration after cordocentesis. A second ultrasound examination at 27 wk gestation showed further enlargement of the goiter (34/21 mm). Two intraamniotic injections of 200 microg levothyroxine were performed during the seventh month of pregnancy. Ultrasound studies revealed a fetal goiter size of 30/18 mm during the eighth month of gestation. The woman delivered at term a female infant with an Apgar score of 10 at 1 and 5 min. Cord blood analysis indicated elevated TSH (284 mU/liter) and low free T(4) (5.5 pmol/liter) levels. The serum thyroglobulin (Tg) concentration was low (0.8 ng/ml), whereas ultrasound of the neonate indicated an enlarged thyroid gland (32/15/14 mm). During the second pregnancy, ultrasound examination revealed a goiter, and fetal hypothyroidism was also confirmed after umbilical vein blood sampling (TSH, 472 mU/liter). After two intraamniotic injections of 500 microg levothyroxine, the woman delivered a male infant at 37 wk of pregnancy. In cord blood the serum TSH concentration was 39 mU/liter, and the serum Tg level was low (0.7 ng/ml). The parents were nonconsanguineous. After birth of the two affected siblings, genomic DNA sequencing identified the presence of compound heterozygous mutations of the Tg gene: the paternal mutation consists of a cytosine deletion at nucleotide 1143 in exon 9 (1143delC), resulting in a frameshift that generates a stop codon at position 382, and the maternal mutation is a guanine to adenine substitution at position 6725 in exon 38, creating the R2223H missense mutation in the acetylcholinesterase homology domain of Tg. In conclusion, we report two siblings with congenital goiter and hypothyroidism caused by compound heterozygous mutations of the Tg gene.

Adult↗

PubMed2002

Genotyping and characterization of two polymorphic microsatellite markers located within introns 29 and 30 of the human thyroglobulin gene.

The purpose of the present work was to characterize two new polymorphic microsatellite markers in the thyroglobulin gene. TGrI29 and TGrI30 repeats are located within introns 29 and 30, respectively. Genetic studies were carried out by using polymerase chain reaction (PCR) followed by denaturing polyacrilamide gel electrophoresis. TGrI29 exhibited clearly 4 distinguishable alleles ranging from 197 to 203 base pair (bp) in length and TGrI30 showed 8 alleles ranging from 502 to 542 bp. We characterized the two markers by determinating allele frequencies and measures of variation. The heterozygosities (HET) observed of TGrI29 and TGrI30 were 0.859 and 0.522, respectively. The polymorphism information contents (PIC) were 0.471 and 0.434, respectively. No significant differences from Hardy-Weinberg values were found for these two systems. The PCR products of each allele were cloned using the pGEM-T Easy vector and directly sequenced by Taq polymerase-based chain terminator method. Sequencing analysis indicated that both loci are complex repeats, TGrI29 containing two types of variable motifs (tc)n and (tg)n, and TGrI30 a tetra-nucleotide tandem units (atcc)n. In two TGrI29 alleles and one TGrI30 allele were found two different subtypes in each one, with the same molecular weights but different distribution of the tandem repeats. In conclusion, both microsatellites analyzed are highly informative polymorphic markers and can be used in linkage studies in families with congenital hypothyroidism or autoimmunity thyroid diseases.

Amino Acid Sequence↗