Correlation between B-RAFV600E mutation and clinico-pathologic parameters in papillary thyroid carcinoma: data from a multicentric Italian study and review of the literature.

Recently, a somatic point mutation of the B-RAF gene (V600E) has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence variable among different series. Since discordant data on the clinico-pathologic features of B-RAF mutated PTC are present in the literature, the aim of the present co-operative study was to establish the prevalence of this genetic alteration and to perform a genotype-phenotype correlation in a large cohort of patients with PTC. To this purpose, a series of 260 sporadic PTCs with different histological variants were included in the study. The mutational analysis of the B-RAF gene was performed either by RT-PCR followed by single-stranded conformational polymorphism or by PCR and direct sequencing. Statistical analyses were obtained by means of chi2/Fisher's exact test and t-test. Overall, a heterozygous T > A transversion at nucleotide 1799 (V600E) was found in 99 out of 260 PTCs (38%). According to the histological type of the tumor, the B-RAF (V600E) mutation was present in 48.3% of cases of classic PTCs (85 out of 176), in 17.6% (nine out of 51) of follicular variants of PTCs, in 21.7% (five out of 23) in other PTC variants and in none of the ten poorly differentiated tumors. B-RAF (V600E) was significantly associated with the classic variant of PTC (P = 0.0001) and with an older age at diagnosis (P = 0.01). No statistically significant correlation was found among the presence of B-RAF (V600E) and gender, tumor node metastasis (TNM), multicentricity of the tumor, stage at diagnosis and outcome. In conclusion, the present study reports the prevalence of B-RAF (V600E) (38%) in the largest series of sporadic PTCs, including 260 cases from three different Italian referring centers. This prevalence is similar to that calculated by pooling together all data previously reported, 39.6% (759 out of 1914 cases), thus indicating that the prevalence of this genetic event lies around 38-40%. Furthermore, B-RAF (V600E) was confirmed to be associated with the papillary growth pattern, but not with poorer differentiated PTC variants. A significant association of B-RAF mutation was also found with an older age at diagnosis, the mutation being very rare in childhood and adolescent PTCs. Finally, no correlation was found with a poorer prognosis and a worse outcome after a median follow-up of 72 months.

Syndromes of thyroid hormone resistance.

Thyroid hormone resistance (RTH) is a rare autosomal dominant disorder, characterized clinically by goiter and biochemically by elevated circulating free thyroid hormone levels in the presence of measurable serum TSH concentrations. About 85% of patients with RTH are harboring mutations in thyroid hormone receptor beta (TRB). These mutations cluster in three different "hot spot" in the T3 binding domain of the receptor. When mapped to their homologous residues in the TR crystal structure, these three clusters of mutations border the T3-binding pocket. As a consequence, most TRB mutations impair the hormone binding to the receptor and interfere with the mechanism(s) of corepressor release and the consequent recruitment of coactivators. Thus, the remodeling of chromatin structure throughout the process of histone acetylation is prevented and the transcriptional activity of the mutant receptor on both positively and negatively regulated genes, severely disrupted. The lack of interaction with coactivators appears to be an additional mechanism for the dominant negative effects of mutant TRB on the transcriptional activity of the normal receptor.

A novel splice variant involving the 5' untranslated region of thyroid hormone receptor beta1 (TRbeta1).

The thyroid hormone receptor beta (TRbeta) gene generates two different proteins by use of a different promoter (beta1 and beta2). We now report a novel short TRbeta1 RNA splice variant in humans lacking 35 nucleotides at the 3' end of the non-coding exon 1 due to an alternative 5' splice donor site. This short variant was first identified in sequences of cDNA obtained from cultured human fibroblasts. Both variants were found in human fibroblasts, brain, pituitary, adrenal gland, placenta, muscle, thyroid and lymphocytes. These TRbeta1 variants possess splice donor sites with a sequence score slightly favoring the TRbeta1 long variant. Variant-specific real-time polymerase chain reaction (PCR) showed that their relative proportions were equal except in pituitary and muscle, in which the long form was 3- and 5-fold in excess. T3 treatment of fibroblasts grown in thyroid hormone depleted medium did not affect the absolute or relative expression of the two variants. Furthermore, the expression level in fibroblasts from patients with resistance to thyroid hormone with or without TRbeta gene mutations was not different to that in fibroblasts from normal controls.

Monoallelic expression of mutant thyroid peroxidase allele causing total iodide organification defect.

Mutations in the thyroid peroxidase (TPO) gene lead to severe congenital hypothyroidism due to total iodide organification defect (TIOD). According to the recessive mode of inheritance, patients are homozygous or compound heterozygous for gene mutations. However, about 17% of cases with typical phenotype harbor a single TPO-mutated allele. We present a TIOD family in which the three affected siblings had a single genomic TPO mutation (R693W) inherited from the unaffected father. Other mutations were not found, although all TPO coding exons and exon/intron boundaries were sequenced. Eleven different polymorphisms were found in hetero- or homozygosity in all family members. On the contrary, using retrotranscribed thyroid tissue RNA, all heterozygous polymorphisms and the mutation were homozygous. The distribution of the polymorphisms indicated that only the mutant paternal allele is transcribed at the thyroid tissue level. We excluded the presence of major deletions involving the maternal chromosome at 2p25 and of maternal imprinting or mutations in part of the regulatory regions of the gene. In summary, we report one family with TIOD due to monoallelic expression of a mutant TPO allele in the thyroid. This mechanism might be generally involved in TIOD cases with a single TPO-mutated allele.

Genetic analyses and evaluation of peripheral parameters of thyroid hormone action for the differential diagnosis of RTH. A novel heterozygous missense mutation (M334T) discovered.

Resistance to thyroid hormone (RTH) is a rare disease characterized by goiter and elevated free thyroid hormone (TH) levels in the presence of detectable concentrations of TSH. Most RTH patients harbor mutations in the ligand binding domain (LBD) of thyroid hormone receptor beta (TRbeta) gene, without a clear correlation between genotype and phenotype. Clinical, biochemical and genetic analyses were performed in several members of one family, because the index case presented with elevated free TH, measurable TSH and no hyperthyroid manifestations, but with a pituitary lesion at MRI. High free TH levels and TSH concentrations in the normal range were found also in 4 relatives. The presence of euthyroidism in all patients together with peripheral parameters of TH action in the normal range led to the diagnosis of generalized RTH (GRTH). In the five affected members, the genetic analysis revealed a novel heterozygous missense mutation at codon 334 (M334T). A different mutation at codon 334 was previously described in association with selective pituitary resistance to thyroid hormone (PRTH). Therefore, we confirm that substitutions at Methionine 334 are critical for the structural integrity of TRbeta LBD. The association of different phenotypes with substitutions affecting the same codon is another contribution confirming that RTH phenotype does not generally depend upon the site of the mutation in the LBD of TRbeta1.

Multigenerational familial medullary thyroid cancer (FMTC): evidence for FMTC phenocopies and association with papillary thyroid cancer.

BACKGROUND: Occurrence in a familial setting is well established for medullary thyroid carcinoma (MTC) and has been more recently reported for papillary thyroid cancer (PTC). Germline mutations or rearrangements of the RET proto-oncogene are the genetic background of the majority of hereditary MTCs and of about 25-40% of PTCs. PATIENTS: A large multigenerational familial medullary thyroid cancer (FMTC) family, comprised of four generations and a total of 60 subjects, has been fully evaluated. Studies on germline RET mutations and polymorphisms, on somatic RET activation and on haplotyping with RET-linked markers, were performed. RESULTS: RET mutational analysis revealed a rare missense point mutation in exon 15 of RET (A891S), associated with FMTC. Haplotype analysis showed a co-segregation between the allelic variant 5 of D10S578 marker (which is tightly linked to the RET locus) and the RET mutation. Two patients, from different branches of the family, did not harbour the point mutation A891S despite histological confirmation of MTC. In these cases, haplotype analysis excluded the involvement of the RET gene itself in the pathogenesis of the MTC. In three patients, the coexistence, in different foci, of medullary and papillary thyroid cancer was documented. The genetic studies did not show ret/PTC rearrangements. The microsatellite analysis excluded co-segregation of RET locus with the MTC/PTC phenotype. CONCLUSIONS: We report a full clinical and molecular analysis of a large FMTC kindred with an uncommon RET mutation. In two family members, phenotype and genotype were not concordant, representing the first evidence of FMTC phenocopies. Furthermore, the association of familial forms of medullary and papillary thyroid cancers has been found in 30% of patients undergoing thyroidectomy for MTC. In these situations, genetic analyses excluded the possible germline involvement of RET. Though FMTC phenocopies are likely to represent an exceptional finding, such a possibility should be taken into account in the genetic counselling for MEN 2 syndromes.

The role of pendrin in iodide regulation.

Recent advances in human genetics have catalyzed the attention on Pendred's syndrome and its disease-gene, PDS. Studies on the expression of the PDS gene and on the function of its encoded protein, which has been named pendrin, are currently in progress. Consistent with the Pendred's syndrome phenotype, which is characterized by thyroid dysfunction associated to deafness, PDS expression has been demonstrated in the thyroid and in the inner ear. Despite its high homology to known sulfate transporters, pendrin has been shown to transport iodide and chloride, but not sulfate. Thus, it is probably devoted to regulate, at the apical membrane where it has been immunolocalized, the flux of iodide from the thyroid cell to the colloid space. The function of pendrin in the inner ear is not well understood, but it seems to function also at this level as an anion transporter. Indeed, a pronounced PDS expression has been detected in structures of the inner ear, such as the membranous labyrinth and the endolymphatic duct and sac. At this level, the possible role of pendrin could be the maintenance of the appropriate ionic composition of the endolymph. Although many questions remain to be answered, these recent achievements concerning the putative role of pendrin aid to better understand the genetic basis of the peculiar phenotype of Pendred's syndrome, which associate the dysfunction of two so different organs such as the thyroid and the inner ear.

Molecular analysis of the Pendred's syndrome gene and magnetic resonance imaging studies of the inner ear are essential for the diagnosis of true Pendred's syndrome.

Pendred's syndrome is a combination of congenital sensorineural hearing loss and iodine organification defect leading to a positive perchlorate test and goiter. Although it is the commonest form of syndromic hearing loss, the variable clinical presentation contributes to the difficulty in securing a diagnosis. The identification of the disease gene (PDS) prompts the need to reevaluate the syndrome to identify possible clues for the diagnosis. To this purpose, in three Italian families presenting with the clinical features of Pendred's syndrome, the molecular analysis was accompanied by full clinical, biochemical, and radiological examination. A correlation between genotype and phenotype was found in the only patient with enlargement of vestibular aqueduct and endolymphatic duct and sac at magnetic resonance imaging. This subject was a compound heterozygote for a deletion in PDS exon 10 (1197delT, FS400) and a novel insertion in exon 19 (2182-2183insG, Y728X). The present study demonstrates for the first time the value of the combination of clinical/radiological and genetic studies in the diagnosis of Pendred's syndrome. The positivity of a perchlorate discharge test and the malformations of membranous labyrinth fit well with the recent achievements on the role of pendrin in thyroid hormonogenesis and the maintenance of endolymph homeostasis.

Prenatal diagnosis of thyroid hormone resistance.

A 29-yr-old woman with pituitary resistance to thyroid hormones (PRTH) was found to harbor a novel point mutation (T337A) on exon 9 of the thyroid hormone receptor beta (TRbeta) gene. She presented with symptoms and signs of hyperthyroidism and was successfully treated with 3,5,3'-triiodothyroacetic acid (TRIAC) until the onset of pregnancy. This therapy was then discontinued in order to prevent TRIAC, a compound that crosses the placental barrier, from exerting adverse effects on normal fetal development. However, as the patient showed a recurrence of thyrotoxic features after TRIAC withdrawal, we sought to verify, by means of genetic analysis and hormone measurements, whether the fetus was also affected by RTH, in order to rapidly reinstitute TRIAC therapy, which could potentially be beneficial to both the mother and fetus. At 17 weeks gestation, fetal DNA was extracted from chorionic villi and was used as a template for PCR and restriction analysis together with direct sequencing of the TRbeta gene. The results indicated that the fetus was also heterozygous for the T337A mutation. Accordingly, TRIAC treatment at a dose of 2.1 mg/day was restarted at 20 weeks gestation. The mother rapidly became euthyroid, and the fetus grew normally up to 24 weeks gestation. At 29 weeks gestation mild growth retardation and fetal goiter were observed, prompting cordocentesis. Circulating fetal TSH was very high (287 mU/L) with a markedly reduced TSH bioactivity (B/I: 1.1 +/- 0.4 vs 12.7 +/- 1.2), while fetal FT4 concentrations were normal (8.7 pmol/L; normal values in age-matched fetuses: 5-22 pmol/L). Fetal FT3 levels were raised (7.1 pmol/L; normal values in age-matched fetuses: <4 pmol/L), as a consequence of 100% cross-reactivity of TRIAC in the FT3 assay method. To reduce the extremely high circulating TSH levels and fetal goiter, the dose of TRIAC was increased to 3.5 mg/day. To monitor the possible intrauterine hypothyroidism, another cordocentesis was performed at 33 weeks gestation, showing that TSH levels were reduced by 50% (from 287 to 144 mU/L). Furthermore, a simultaneous ultrasound examination revealed a clear reduction in fetal goiter. After this latter cordocentesis, acute complications occured, prompting delivery by cesarean section. The female neonate was critically ill, with multiple-organ failure and respiratory distress syndrome. In addition, a small goiter and biochemical features ofhypothyroidism were noted transiently and probably related to the prematurity of the infant. At present, the baby is clinically euthyroid, without goiter, and only exhibits biochemical features of RTH. In summary, although further fetal studies in cases of RTH are necessary to determine whether elevated TSH levels with a markedly reduced bioactivity are a common finding, our data suggest transient biochemical hypothyroidism in RTH during fetal development. Furthermore, we advocate prenatal diagnosis of RTH and adequate treatment of the disease in case of maternal hyperthyroidism, to avoid fetal thyrotrope hyperplasia, reduce fetal goiter, and maintain maternal euthyroidism during pregnancy.

Clinical and hormonal outcome after two years of triiodothyroacetic acid treatment in a child with thyroid hormone resistance.

We report here a new family with thyroid hormone resistance (RTH), with phenotypic variability among subjects. Particular emphasis is given to the clinical and hormonal outcome after 2 years of triiodothyroacetic acid (TRIAC) treatment in an affected child with peripheral thyrotoxic features (pituitary RTH [PRTH]). The genetic defect was a substitution in position 1642 (C to A) within the exon 10 of thyroid hormone receptor beta1 (TRbeta1) gene, resulting in the codon change P453T. The mutant receptor had a significantly reduced triiodothyronine (T3) binding affinity. Within this family, the child and the mother suffered from hyperthyroidism and were clinically classified as PRTH, while the maternal grandmother was clinically euthyroid, indicating a generalized form of the disease (GRTH). Rapid normalization of heart rate was initially obtained by the association of the cardioselective beta-blocker atenolol with TRIAC. Nevertheless, long-term TRIAC therapy, through its lowering action of serum thyrotropin (TSH) and thyroid hormone levels, maintained a normal heart rate after atenolol discontinuation and normalized the neurological disturbances and the clinical signs in the child, without any apparent side effect. In fact, growth velocity remained unchanged and no alteration of several parameters of thyroid hormone action at the tissue level was observed, whereas soluble interleukin-2 receptor levels improved significantly, confirming the safety and efficacy of long-term TRIAC therapy for PRTH also during childhood. We thus recommend testing the efficacy of TRIAC therapy in all RTH patients presenting with clinical features of hyperthyroidism.

Thyrotropin-secreting pituitary tumors in hyper- and hypothyroidism.

Thyrotropin (TSH)-secreting pituitary tumors may be found in two opposite clinical situations: the hyperthyroidism secondary to thyrotroph adenomas, also called central hyperthyroidism, and the long-standing primary hypothyroidism which can be accompanied by a compensatory pituitary enlargement. TSH-secreting pituitary adenomas belong to the syndromes of "inappropriate secretion of TSH" (IST). The adjective "inappropriate" indicates the lack of the expected suppression of TSH secretion when free thyroid hormone levels are actually elevated, as in the other forms of thyrotoxicosis. Moreover, TSH-omas have to be differentiated from the non-neoplastic form of IST which is due to resistance to thyroid hormone. Differently, pituitary hyperplasia, which is reversible on thyroid hormone replacement, is the more frequent cause of a pituitary mass occurring in the context of untreated primary hypothyroidism. Failure or delay in the recognition of the above clinical situations may cause dramatic consequences, such as unnecessary pituitary surgery in hypothyroid patients or improper thyroid ablation in those with central hyperthyroidism. In contrast, early diagnosis and proper treatment of TSH-secreting pituitary tumors prevents the appearance of signs and symptoms of mechanical compression of the adjacent structures by the expanding tumor mass (visual field defects, headache and hypopituitarism).