Perinatal mortality in cattle associated with goitre

The role of iodine in ruminant reproductive function is well known. However, studies about the impact of this deficiency in reproductive performance of cattle are scarce. This paper describes stillbirth episodes in three beef cow herds in northwest Argentina. The stillbirth losses ranged between 15.8 and 25%. Grossly, the thyroid glands presented diffuse, bilateral and symmetric enlargement. Microscopically, follicular epithelium showed severe hyperplasia and hypertrophy, forming multiple stratified layers of large cuboidal cells, with papillary projections into the follicular lumen. Free thyroxine (T4f) mean levels in serum from aborted cows and heifers was 0.63±0.05 ng/dl. A diagnosis of congenital goiter was performed based on these findings. Following the diagnosis, the herds were supplemented orally and parentally with iodine. Mean T4f level post-treatment was 0.80±0.05 ng/dl and the stillbirth rate dropped sharply.(AU)

Perinatal mortality in cattle associated with goitre

The role of iodine in ruminant reproductive function is well known. However, studies about the impact of this deficiency in reproductive performance of cattle are scarce. This paper describes stillbirth episodes in three beef cow herds in northwest Argentina. The stillbirth losses ranged between 15.8 and 25%. Grossly, the thyroid glands presented diffuse, bilateral and symmetric enlargement. Microscopically, follicular epithelium showed severe hyperplasia and hypertrophy, forming multiple stratified layers of large cuboidal cells, with papillary projections into the follicular lumen. Free thyroxine (T4f) mean levels in serum from aborted cows and heifers was 0.63±0.05 ng/dl. A diagnosis of congenital goiter was performed based on these findings. Following the diagnosis, the herds were supplemented orally and parentally with iodine. Mean T4f level post-treatment was 0.80±0.05 ng/dl and the stillbirth rate dropped sharply.

Novel compound heterozygous Thyroglobulin mutations c.745+1G>A/c.7036+2T>A associated with congenital goiter and hypothyroidism in a Vietnamese family. Identification of a new cryptic 5' splice site in the exon 6.

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.

Clinical, biochemical, and molecular findings in Argentinean patients with goitrous congenital hypothyroidism.

We describe the clinical, biochemical, and molecular findings of a cohort of Argentinean patients with congenital hypothyroidism (CH) and goiter studied to characterize iodide organification and thyroglobulin (TG) defects. 20 CH patients (16 unrelated) were grouped according to serum TG levels and a perchlorate discharge test (PDT) in: group 1 (G1): nine patients with high TG and PDT > 10% who were studied for tiroperoxidase (TPO), dual oxidase 2 (DUOX2), and dual oxidase A2 (DUOXA2) defects and group 2 (G2): 11 patients with low TG and PDT < 10% studied for TG defects. Goiter characteristics, outcome, and TT4 and TT3 levels without treatment were compared between groups. 6/9 G1 patients harbored mutations in TPO gene and 3/9 in DUOX2 gene. In G2, mutations of TG gene were found in 3/11 homozygous, 5/11 compound heterozygous, and 3/11 heterozygous patients. Goiter was only evidenced by thyroid scan in the neonatal period in both groups; was moderately enlarged in patients diagnosed during infancy. In the late detected patients, goiter was big and nodular in G1 while diffuse and moderate in G2. Early detected patients grew and developed normally while those diagnosed late were severely mentally retarded in G1 and only mildly retarded in G2. Thyroid hormone levels of G1 were significantly lower than those of G2 P < 0.01. Molecular approach to characterize defects in organification and TG defects was optimized by TG measurements and PDT. Clinical and biochemical differences based on molecular findings will allow further investigations on genotype-phenotype relationships.

Non-immune goiter and hypothyroidism in a 19-week fetus: a plea for conservative treatment.

Hypothyroidism was documented by cordocentesis at 19 weeks in a fetus with non-immune goiter. Intra-amniotic thyroxine was injected at 25 weeks when amniotic fluid volume increased. Psychomotor outcome was normal. We argue that intra-amniotic thyroxine should not be used to treat the hypothyroidism but only to correct the development of polyhydramnios.

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.

Resistencia a Hormonas Tiroideas (RHT): Descripción de una nueva mutación / Resistance to Thyroid Hormones (RTH): Description of a new mutation

Introducción: La resistencia a hormonas tiroideas (RHT) es un desorden genético de transmisión dominante poco frecuente, caracterizado por una respuesta reducida de los tejidos blanco a las hormonas tiroideas. RHT está ligada al gen del receptor beta de hormona tiroidea (TRβ). El síndrome se identifica por niveles persistentemente elevados de T4 y T3 totales y libres en presencia de TSH no suprimida. Materiales y Métodos: Paciente femenina de 62 años de edad con antecedente de hemitiroidectomía a los 22 años por bocio. Clínicamente, la mujer se encontraba eutiroidea y hemodinámicamente estable. En los exámenes complementarios se constató la presencia de nódulo tiroideo, con estudio citológico benigno y en el laboratorio hormonas tiroideas totales y libres elevadas con TSH no suprimida. La impresión diagnóstica fue RHT, siendo el principal diagnóstico diferencial el tirotropinoma. Se realizó perfil tiroideo completo en el caso índice y en dos familiares de primer grado. Se dosaron gonadotropinas y prolactina, y se realizó RMN de hipófisis en el caso índice. Se estudiaron mutaciones del gen TRβ en ADN genómico en la paciente y en uno de sus familiares. Resultados: Avalando la impresión diagnóstica, tanto el caso índice como los dos familiares mostraron un perfil tiroideo compatible con RHT. El estudio genético identificó una nueva mutación en el exón 10: c.1339C>A que resulta en una sustitución p.P447T. La misma fue observada tanto en el caso índice como en el familiar estudiado. Conclusión: La historia de esta paciente con RHT, al igual que otros casos descriptos en la bibliografía, remarcan la importancia de un diagnóstico adecuado y temprano de esta patología poco frecuente para evitar conductas terapéuticas iatrogénicas y con consecuencias relevantes en la vida de estos pacientes. Paralelamente, se describe una nueva mutación genética en esta familia.

Introduction: Resistance to thyroid hormones (RTH) is an unusual autosomal dominant inherited disorder characterized by a reduced target organ responsiveness to thyroid hormones. RTH is linked to the gene encoding the thyroid receptor β (TR β). This syndrome is characterized by persistent high levels of total and free T4 and T3 while TSH is not inhibited. Materials and Methods: 62 years old female who underwent a partial thyroidectomy because of goiter forty years ago. Clinically, she seemed to be an euthyroid patient and her hemodynamic status was normal. The exams revealed the existence of a benign thyroid nodule, high levels of total and free thyroid hormones and normal values of TSH. Our diagnostic impression was RTH, though differential diagnosis with thyrotropin secreting pituitary adenoma was mandatory. Complete assays of thyroid hormones were performed in the patient and in two first degree relatives. Basal LH, FSH and prolactin were assayed in the patient; and a magnetic resonance imaging of her pituitary gland was obtained. Finally we performed genetic testing in patient's DNA and a relative's DNA to demonstrate gene defect. Results: According to our diagnostic impression, not only the patient's laboratory was compatible with RTH, but so was the laboratory of the two relatives. DNA mutation analisys demonstrated a new mutation in exon 10: c.1339C>A responsible for the substitution p.P447T. This mutation was found in DNA of the patient and DNA of her relative. Conclusion: This patient with RTH, as well as other reported cases, reminds us about the importance of a certain and early diagnosis of this rare disorder in order to avoid iatrogenic treatments. A new mutation is described in this family.

Aspectos patogênicos e clínicos do bócio multinodular / Pathogenic and clinical aspects of multinodular goiter

O bócio multinodular (BMN) é definido como um aumento da glândula tireóide secundário à proliferação multifocal de tireócitos e caracteriza-se pela heterogeneidade no crescimento e função das células foliculares. O BMN é considerado uma neoplasia benigna da tireóide. É uma doença comum, com aumento da prevalência em áreas com deficiência de iodo, sendo este o principal fator etiológico ambiental. A patogênese desta disfunção tireoidiana ainda não está inteiramente elucidada. Nesta revisão serão abordados os principais mecanismos envolvidos na patogênese, seguidos das implicações clínicas dessa patologia.

Multinodular goiter (MNG) is defined as an enlargement of the thyroid gland that is characterized by heterogeneity in growth and function of thyroid follicular cells. MNG is now considered a true thyroid neoplasm. It is a common disease, with higher prevalences in iodine deficiency areas. Iodine deficiency is the main environmental etiologic factor for MNG. The pathogenesis of multinodular goiter is not yet fully clarified. The purpose of this review is to summarize the current knowledge of MNG with respect to the pathology, etiologic and clinical characteristics.

Pendred syndrome in a large consanguineous Brazilian family caused by a homozygous mutation in the SLC26A4 gene / Síndrome de Pendred causada por mutação em homozigoze no gene SLC26A4 em uma família brasileira consangüínea

Pendred Syndrome (PS) is an autossomal recessive disorder characterized by sensorineural deafness, goiter and iodide organification defect. The hearing loss is associated with inner ear abnormalities, ranging from an isolated enlarged vestibular aqueduct (EVA) to a typical coclear dysplasia. Mutations in the gene that encodes pendrin (SLC26A4), a chloride/iodide transporter, have been shown to be associated with PS. We describe the clinical and molecular characteristics of a large consanguineous family harboring a mutation in the SLC26A4 gene. The proband was a 26-year-old deaf Brazilian woman who presented a bulky multinodular goiter and hypothyroidism since puberty. Five other siblings were deaf: one brother had a similar phenotype, three siblings also had goiters but normal thyroid function tests, and one brother had only a subtle thyroid enlargement. Other 4 siblings had no thyroid or hearing disorder. Parents were first degree cousins and had normal hearing. The mother was healthy, except for subclinical hypothyroidism; the father was deceased. A perchlorate test in the proband showed a discharge of 21 percent of the incorporated iodide 2h after the administration of 1g of KClO4. Audiological examinations showed profound hearing loss in all deaf subjects; CT and MRI of the temporal bones showed EVA in all of them. Genomic DNA was isolated from whole blood, from the 6 affected and 4 unaffected siblings, the mother and control. The coding region of the PDS gene (exons 2-21), including exon/intron boundaries, were amplified by PCR and sequenced. A single base-pair (T) deletion at position 1197 of exon 10 was detected in homozygous state in the 6 deaf siblings. The mother and 2 unaffected siblings were heterozygous for this mutation, which has been described by Everett et al. The 1197delT mutation is predicted to result in a frameshift and a truncated protein. The existence of PS phenocopies and intrafamilial phenotypic variability are...

A syndrome de Pendred (SP) é uma doença autossômica recessiva caracterizada por surdez neurossensorial, bócio e defeito de organificação do iodo. A perda auditiva está associada a anormalidades do ouvido interno, desde a dilatação isolada do aqueduto vestibular (DAV) até uma típica displasia coclear. Mutações no gene que codifica a pendrina (SLC26A4), um transportador de cloreto/iodeto, têm sido associadas à SP. Descrevemos as características clínicas e moleculares de uma grande família consangüínea portadora de uma mutação no gene SLC26A4. O caso-índice era uma paciente do sexo feminino, brasileira, 26 anos, portadora de surdez congênita, que apresentava um volumoso bócio multinodular e hipotireoidismo desde a puberdade. Outros cinco irmãos eram surdos: um irmão tinha fenotipo semelhante, três também tinham bócio, porém com função tiroideana normal e um irmão tinha apenas um discreto aumento da tiróide. Outros quatro irmãos não apresentavam alteração tiroideana ou auditiva. Os pais eram primos de primeiro grau e tinham audição normal. A mãe era saudável, exceto por hipotireoidismo subclínico; o pai era falecido. O teste do perclorato no caso-índice revelou a liberação de 21 por cento do iodo incorporado duas horas após a administração de 1 g de KClO4. Os exames audiológicos mostraram perda auditiva profunda em todos os indivíduos afetados; TC e RMN dos ossos temporais mostraram DAV em todos eles. O DNA genômico foi isolado do sangue total dos seis irmãos afetados e dos quatro não-afetados, da mãe e do controle. A região codificante do gene PDS (éxons 2-21), incluindo as junções éxon/íntron, foram amplificadas por PCR e seqüenciadas. Foi detectada a deleção de uma base (T) na posição 1197 do éxon 10, em homozigoze, nos seis irmãos afetados. A mãe e dois irmãos não-afetados eram heterozigotos para a mutação, que foi descrita inicialmente por Everett e cols. A mutação 1197delT provavelmente resulta em um erro de fase de leitura (frameshift)...

Pendred syndrome in a large consanguineous Brazilian family caused by a homozygous mutation in the SLC26A4 gene.

Pendred Syndrome (PS) is an autossomal recessive disorder characterized by sensorineural deafness, goiter and iodide organification defect. The hearing loss is associated with inner ear abnormalities, ranging from an isolated enlarged vestibular aqueduct (EVA) to a typical coclear dysplasia. Mutations in the gene that encodes pendrin (SLC26A4), a chloride/iodide transporter, have been shown to be associated with PS. We describe the clinical and molecular characteristics of a large consanguineous family harboring a mutation in the SLC26A4 gene. The proband was a 26-year-old deaf Brazilian woman who presented a bulky multinodular goiter and hypothyroidism since puberty. Five other siblings were deaf: one brother had a similar phenotype, three siblings also had goiters but normal thyroid function tests, and one brother had only a subtle thyroid enlargement. Other 4 siblings had no thyroid or hearing disorder. Parents were first degree cousins and had normal hearing. The mother was healthy, except for subclinical hypothyroidism; the father was deceased. A perchlorate test in the proband showed a discharge of 21% of the incorporated iodide 2h after the administration of 1g of KClO4. Audiological examinations showed profound hearing loss in all deaf subjects; CT and MRI of the temporal bones showed EVA in all of them. Genomic DNA was isolated from whole blood, from the 6 affected and 4 unaffected siblings, the mother and control. The coding region of the PDS gene (exons 2-21), including exon/intron boundaries, were amplified by PCR and sequenced. A single base-pair (T) deletion at position 1197 of exon 10 was detected in homozygous state in the 6 deaf siblings. The mother and 2 unaffected siblings were heterozygous for this mutation, which has been described by Everett et al. The 1197delT mutation is predicted to result in a frameshift and a truncated protein. The existence of PS phenocopies and intrafamilial phenotypic variability are well documented. The definite diagnosis requires molecular analysis. Our study illustrates the value and challenges of mutational analysis in selected patients with PS.

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.

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).

Thyroperoxidase gene mutations in congenital goitrous hypothyroidism with total and partial iodide organification defect.

Mutations of the thyroperoxidase (TPO) gene have been reported as being the most severe and frequent abnormality in thyroid iodide organification defect (IOD) causing goitrous congenital hypothyroidism. The objective of this study was to screen and subsequently identify TPO gene mutations in patients with congenital hypothyroidism with evidence of total iodine organification defects (TIOD) or partial iodine organification defect (PIOD) as defined by the perchlorate discharge test. Seven goitrous patients with TIOD and seven patients with PIOD, from three and five unrelated families, respectively, were studied. We were able to detect different TPO genes mutations in patients with TIOD and PIOD. In TIOD families the results were as follows: (1) a homozygous GGCC insertion at exon 8, position 1277 (family 1); (2) compound heterozygosity with a GGCC insertion at exon 8 (1277) and a nucleotide substitution in exon 11 (2068G>C) (family 2); (3) compound heterozygosity with the mutation 2068G>C in exon 11 and a C insertion in exon 14 between positions 2505-2511 (family 3). In patients with PIOD we have detected: (1) only one heterozygous mutation in two families (4 and 5), in exons 11 and 10 (2084G>A and 1780C>A); (2) a compound heterozygous condition in one family (family 6), with mutations, respectively in exons 8 and 10 (1242G>T and 1780C>A); (3) only polymorphisms (family VII) and (4) a heterozygous mutation in the first base of the border exon/intron 9 +1G>T (family VIII). We did not detect inactivating mutations in exons 11, 16, and 21 of the THOX2 gene where mutations have been previously described. We concluded that homozygous and compound heterozygous mutations found in TIOD characterized the autosomal recessive mode of inheritance and will translate a nonfunctional protein or a protein that may not reach the apical membrane. As for PIOD, the majority of the studied kindreds had only heterozygous mutations and/or polymorphisms. It is conceivable that these TPO gene sequence alterations may partially affect the functional state of the translated protein or affect its transport to the apical membrane.

Congenital goiter with hypothyroidism caused by a 5' splice site mutation in the thyroglobulin gene.

In this work we have extended our initial molecular studies of a consanguineous family with two affected goitrous siblings (H.S.N. and Ac.S.N.) with defective thyroglobulin (Tg) synthesis and secretion because of a homozygotic deletion of a fragment of 138 nucleotides (nt) in the central region of the Tg mRNA, identified previously in H.S.N. In order to identify the intron/exon boundaries and to analyze the regions responsible for pre-mRNA processing corresponding to a 138 nt deletion, we performed a screening of a human genomic library. The intron/exon junction sequences were determined from one positive clone by sequencing both strands of the DNA template. The results showed that the deletion mapped between positions 5549 and 5686 of the Tg mRNA and corresponded to exon 30. The positions of the exon limits differed by three nucleotides from the previously reported data obtained from direct sequencing of the deleted reverse transcriptase-polymerase chain reaction fragment from H.S.N. These variations are because the intron/exon junctions in this region were not available at the time when the deletion was first described. The deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shortened by 46 residues. The same 138 nt deletion was observed in reverse transcriptase-polymerase chain reaction studies performed in the thyroid tissues from Ac.S.N. Genomic DNA analysis showed that a G to T transversion was observed at position +1 in the donor site of intron 30. Both affected patients (H.S.N. and Ac.S.N.) are homozygous for the mutation whereas the normal sister (At.S.N.) had a normal allele pattern. The functional consequences of the deletion are related to structural changes in the protein molecule that either could modify the normal routing of the translation product through the membrane system of the cell or could impair the coupling reaction. Probably the mutant Tg polypeptide might be functionally active in the production of thyroid hormone, because in the presence of a normal iodine ingestion (approximately 150 microg/day), Ac.S.N. was able to maintain normal serum levels of total triiodothyronine (T3) associated with relatively low serum total thyroxine (T4) with normal somatic development without signs of brain damage.

A novel mutation in the TPO gene in goitrous hypothyroid patients with iodide organification defect.

OBJECTIVE: To screen and subsequently sequence the TPO gene for mutations in patients with congenital goitre, hypothyroidism and evidence for an organification defect (positive perchlorate discharge test). PATIENTS: We have studied seven hypothyroid and congenitally goitrous patients from three unrelated families. DESIGN AND MEASUREMENTS: We have measured serum thyroid hormone levels, 131I uptake, serum TSH and serum Tg concentrations. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified genomic DNA was used to screen for mutations in the TPO gene. RESULTS: DGGE identified the presence of two frameshift mutations: a GGCC duplication in exon 8 (homozygous in one family and heterozygous in the other family) and a heterozygous insertion of a single nucleotide (C) at position 2505-2511 in exon 14. In addition, we have detected an alteration in exon 11, not yet described in the literature, derived from a single nucleotide substitution of a C to G at position 2008, altering the well-conserved amino acid domain among the peroxidases superfamily. This mutation in exon 11 was present in two families that showed heterozygous mutation for exon 8 or for exon 14. CONCLUSIONS: These results could support the hypothesis for a putative compound heterozygosity pattern in the affected patients. The altered phenotype (goitre and hypothyroidism since birth) seems justifiable in view of the possible inactivating character of this novel mutation in exon 11.

Metastatic thyroid carcinoma arising from congenital goiter due to mutation in the thyroperoxidase gene.

A very large cervical tumor that extended to the upper mediastinum was seen in a newborn after an uneventful pregnancy. The computed axial tomography scan confirmed the presence of a solid mass with precise limits and scattered foci of calcifications situated in the anterolateral region of the neck. The infant underwent thyroidectomy on the seventh day after birth. Pathological examination revealed a follicular carcinoma of the thyroid and probable dyshormonogenetic hyperplastic goiter. At 5 months of age, whole body scans indicated the presence of lung and bone metastases, which were treated with therapeutic doses of radioiodine. Genomic DNA was obtained from the newborn, her parents, her paternal aunt, and her paternal grandparents. Denaturing gradient gel electrophoresis analysis of PCR fragments corresponding to exon 14 of the thyroid peroxidase (TPO) gene indicated the presence of a mutant TPO allele present in the propositus, her father, and her paternal grandmother. Sequencing of the TPO gene demonstrated a mutation resulting from an insertion of a single extra cytosine in a stretch of seven cytosines at positions 2505-2511. The insertion caused a frame shift and a stop signal in exon 16. This sequence would translate into a structurally modified and probably inactive TPO protein. We conclude that the aggressive thyroid metastatic carcinoma arose from a dyshormonogenetic goiter caused by a defective TPO protein.

[Immunohistochemical identification of biochemical defect of dyshormonogenetic goiter]. / Defeito bioquímico de bócio disormonogenético identificado através de estudo imuno-histoquímico.

This is a report of two cases of congenital goiter in which it was possible to indicate, through immunohistochemistry method, the disorder in the hormonal synthesis. One of these cases was of a sixteen-year-old woman, with goiter since childhood who used thyroid hormone replacement for ten years. Pathology showed coloid goiter with follicular adenoma. Immunohistochemistry showed strong mainly apical follicular cell thyroglobulin. Thyroglobulin was not found in the coloid. This finding is compatible with a malfunction of thyroglobulin exocitosis. The reaction for thyroid peroxidase was adequate for the age. The other case was a 13 year-old-boy with goiter and thyroid hormone replacement since the age of 1 year. He presented slight mental and growth retardation. Pathology showed coloid goiter with follicular adenoma. Immunohistochemistry study showed a very weak and diffuse reaction for thyroid peroxidase while the reaction for thyroglobulin was adequate. These findings indicate quantitative defect of thyroid peroxidase.

Evidence for the segregation of three different mutated alleles of the thyroglobulin gene in a Brazilian family with congenital goiter and hypothyroidism.

We have previously reported a Brazilian family with congenital goiter, hypothyroidism, and marked impairment of thyroglobulin (Tg) synthesis. Analysis of the Tg mRNA in the goiter of one of the siblings revealed a cytosine to thymine transition creating a stop codon at position 1510. This point mutation is removed from the majority of Tg mRNA transcripts by the preferential generation in the goiter of a 171 nt deleted Tg mRNA by alternative splicing. The nonsense mutation destroys a TaqI site at this position in the mutant Tg gene. Using polymerase chain reaction (PCR) amplification and TaqI digestion we found that two siblings affected with goiter and hypothyroidism, as well as the father and three siblings with normal thyroid function, are all heterozygous for the nonsense mutation. This implies that an additional mutation must be present in the affected individuals, generating a compound heterozygote genotype. A new polymorphism within the thyroglobulin gene represented by three alleles has been detected. This was documented by the TaqI restriction enzyme and phTgM3 probe hybridization that showed a three allelic polymorphism with fragment sizes of 16.5 kb (allele A), 14.5 kb (allele B) and 11.0 kb (allele C). Segregation analysis of these alleles in the family indicated that the two affected siblings were homozygous for the allele C. In contrast the unaffected father and three other siblings, who carried the nonsense mutation, were heterozygous for alleles B and C. Analysis of the Tg genotypes implies that two additional mutations of the Tg gene must segregate in this family to account for the observed phenotypes.

A 138-nucleotide deletion in the thyroglobulin ribonucleic acid messenger in a congenital goiter with defective thyroglobulin synthesis.

Two siblings (HSN and AcSN) with congenital goitrous hypothyroidism were investigated in terms of clinical, biochemical, and molecular biology. Diagnosis of defective thyroglobulin (Tg) was based on findings of low serum T4, low normal or normal serum T3, a negative percholate discharge test, and the virtual absence of the serum Tg response to challenge by bovine TSH. Only minute amounts of Tg-related antigens were detected by RIA in the goitrous tissue (HSN, 0.82 mg/g, compared to 70-90 mg/g in normal thyroid tissue), as confirmed by sodium dodecyl sulfate-agarose gel electrophoresis that indicated the virtual absence of Tg. The Tg messenger ribonucleic acids (mRNAs) from controls and HSN thyroid tissue were first reverse transcribed and then divided into several portions from positions 57-8448; the resulting complementary DNAs were, in turn, amplified by reverse polymerase chain reaction. The amplification of nucleotides 5165-6048 from control thyroid tissue Tg mRNA showed a fragment of 884 base pairs (bp). In contrast, the fragment present in the HSN was +/- 750 bp and lacked the normal fragment. The sequencing of the smaller fragment revealed that 138 bp were missing between positions 5590-5727 of the HSN Tg mRNA. This deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shorter by 46 residues. A cysteine residue is maintained by the junction between the proximal T from leucine 1831 and the distal GT from cysteine 1877. DNA genomic polymerase chain reaction amplification excludes a deletion in the Tg gene and indicates that the deleted 138-nucleotide sequences lie in the same exon. The functional consequences of the deletion are not entirely clear, but it is conceivable that the excision of this segment of the Tg molecule could affect the protein structure, resulting in its premature degradation, very low colloid storage, and diminished thyroid hormone production rate.