Prolactin and human weight disturbances: A puzzling and neglected association.

Weight gain at the outset of prolactinomas in many women is well documented. Yet, this symptom is absent from the clinical descriptions of the disease in textbooks and reviews. This omission is almost certainly due to the absence of a physiological explanation for the phenomenon, as prolactin is not a recognized fat promoting hormone. In this review we present the clinical evidence for a relationship between prolactin and fat accumulation and address some possible mechanisms involved. We put forward the hypothesis that prolactin is a component of a neuroendocrine program - maternal subroutine - aimed at optimizing the care of the young through the production of milk, promotion of maternal behavior and increase in the metabolic efficiency of the mother. These adaptations can enable her to face the extraordinary metabolic expenses of pregnancy and nursing, especially during times of suboptimal environmental conditions. We emphasize the uniqueness of prolactin in that it is a hormone that is tonically inhibited and which has its major effects on the regulation of an inter-individual (the mother - offspring dyad), rather than an intra-individual, system. This approach opens a window to consider the possibility of external events as regulators of this system. It also allows addressing a variety of hitherto unexplained findings reported in the literature. Examples include: association of prolactinomas with paternal deprivation and with stressful life events; pseudocyesis; acute life event-driven episodes of galactorrhea; episodes of rapid weight gain following a life event; prolactin surges (without associated cortisol surges) following some psychological stresses.

Expression of iodine metabolism genes in human thyroid tissues: evidence for age and BRAFV600E mutation dependency.

CONTEXT: Children present a higher susceptibility to developing thyroid cancer after radioiodine exposure and also a higher frequency of functional metastases than adults. OBJECTIVE: To assess the mRNA expression of the sodium/iodide (Na(+)/I(-)) symporter (NIS), the Pendred syndrome gene (PDS), thyroperoxidase (TPO), thyroglobulin (Tg) and TSH receptor (TSH-R) in normal thyroid tissues (NTTs) and papillary thyroid carcinomas (PTCs) among different age groups. METHODS: Analysis included 59 samples: 21 NTTs and 38 PTCs, of which 21 were the classic type (CPTC) and 17 the follicular variant (FVPTC). Patients were divided into three age groups: I (n = 16) 5-21 years, II (n = 13) 22-59 years, and III (n = 10) 60-91 years. The relative mRNA expression of the five target genes was determinate by quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Expression of all genes was significantly higher in NTTs than in PTCs, and it was not age dependent in the NTT group. Among PTCs, the mean expression of PDS, TPO and TSH-R was significantly lower in group II than in group I. PDS, TPO and Tg expression was significantly lower in classic PTCs than in FVPTCs. The difference was related to a higher frequency of the BRAF(V600E) mutation in the former group. CONCLUSIONS: The finding of higher PDS, TPO and TSH-R mRNA expression in paediatric vs. adult primary tumour tissues supports the hypothesis that this might contribute to the increased functional activity of metastases in the paediatric group. The finding that mRNA expression of the target genes in NTT was not age dependent does not provide an explanation for the higher susceptibility in the paediatric group.

Familial non-medullary thyroid carcinoma (FNMTC): analysis of fPTC/PRN, NMTC1, MNG1 and TCO susceptibility loci and identification of somatic BRAF and RAS mutations.

Linkage analysis has identified four familial non-medullary thyroid carcinoma (FNMTC) susceptibility loci: fPTC/PRN (1p13.2-1q22), NMTC1 (2q21), MNG1 (14q32) and TCO (19p13.2). To date, there is no evidence for the involvement of genes from the RAS/RAF signalling pathway in FNMTC. The aim of our study was to evaluate the role of the four susceptibility loci, and RAS/RAF signalling pathway genes, in FNMTC. In total, 8 FNMTC families, and 27 thyroid lesions from family members (22 papillary thyroid carcinomas (PTCs): 11 classic, 10 of the follicular variant and 1 of the mixed variant; 4 follicular thyroid adenomas (FTAs) and 1 nodular goitre (NG)), were evaluated for the involvement of the four susceptibility regions, using linkage and loss of heterozygosity (LOH) analyses. BRAF and H-, N- and K-RAS mutations were also screened in the 27 lesions and patients. Linkage analysis in seven informative families showed no evidence for the involvement of any of the four candidate regions, supporting a genetic heterogeneity for FNMTC. Twenty tumours (74%), of which 18 were PTCs, showed no LOH at the four susceptibility loci. The remaining seven tumours (four PTCs, two FTAs and one NG) showed variable patterns of LOH. Fourteen tumours (52%) had somatic mutations: BRAF-V600E mutation was observed in 9 out of the 22 PTCs (41%); and H-RAS and N-RAS mutations were detected in 5 out of the 22 PTCs (23%). Our data suggest that the four candidate regions are not frequently involved in FNMTC and that the somatic activation of BRAF and RAS plays a role in FNMTC tumourigenesis.

Mapping a new familial thyroid epithelial neoplasia susceptibility locus to chromosome 8p23.1-p22 by high-density single-nucleotide polymorphism genome-wide linkage analysis.

CONTEXT: Familial nonmedullary thyroid carcinoma (FNMTC) accounts for approximately 5% of all thyroid tumors. Genetic mapping studies have identified four different chromosomal regions predisposing to FNMTC: fPTC/PRN (1p13.2-1q22), NMTC1 (2q21), MNG1 (14q32), and TCO (19p13.2). OBJECTIVE: Our objective was to map the gene predisposing to familial thyroid epithelial neoplasia in a large Portuguese family. METHODS AND RESULTS: The clinical screening of a Portuguese family identified 11 members affected with benign thyroid lesions and five affected with thyroid carcinomas. Linkage analysis excluded the involvement of the fPTC/PRN, NMTC1, MNG1, and TCO loci. To map the gene predisposing to thyroid epithelial neoplasia in this family, a genome-wide linkage analysis was conducted, using DNA samples from 17 family members and high-density single-nucleotide polymorphism arrays. A genome-wide significant evidence of linkage, to a single region on chromosome 8p23.1-p22 was obtained, with a maximum parametric haplotype-based LOD score of 4.41 (theta=0.00). Linkage analysis with microsatellite markers confirmed linkage to 8q23.1-p22, and recombination events delimited the minimal region to a 7.46-Mb span. Seventeen suggestive candidate genes located in the minimal region were excluded as susceptibility genes by mutational analysis. Allelic losses in the 8p23.1-p22 region were absent in seven thyroid tumors from family members, suggesting that the inactivation of a putative tumor suppressor gene may have occurred through other mechanisms. CONCLUSIONS: Our results present evidence for the existence of a novel familial thyroid epithelial neoplasia susceptibility locus on chromosome 8p23.1-p22, providing the basis for the identification of a gene for this disease.

Mutation analysis of the RET proto-oncogene and early thyroidectomy: results of a Portuguese cancer centre.

BACKGROUND: Evidence that germline mutations in the RET proto-oncogene are the underlying cause of the familial form of medullary thyroid carcinoma (MTC) made it possible to identify gene carriers with a very high degree of accuracy. Aiming to define the mutational profile observed in our patients and to assess gene carriers' compliance with an early surgery, we reviewed results of molecular analysis of RET performed at our institution since 1994. METHODS: One hundred fifty-eight individuals were screened for germline mutations of the RET proto-oncogene. Seventy-seven patients had apparently sporadic MTC; 8 patients had both MTC and pheochromocytoma or MTC and clinical features of multiple endocrine neoplasia type 2B despite a negative family history; 8 patients were known to belong to affected kindreds; and 65 individuals were at-risk individuals to develop MTC. RESULTS: A germline mutation in RET was identified in 4% of apparently sporadic MTC patients, in 100% of patients with MTC and pheochromocytoma or MTC and clinical features of multiple endocrine neoplasia type 2B, and in 100% of probands of clinically established kindreds. The most affected codon was 634 (58%) followed by codon 804 (16%). Among at-risk individuals, 49% were identified as gene carriers. Seven individuals were submitted to prophylactic thyroidectomy (mean age, 17.7 +/- 12.5 years; range: 3-42 years), and all but 1 had MTC. CONCLUSIONS: RET mutational spectrum observed in the present population disclosed a higher frequency of codon 804 mutations than expected. Compliance with an early prophylactic surgery seemed to be influenced not only by medical advice and cultural factors but also by the aggressiveness of disease in gene carriers' families.

The Calcium/Phosphorus Homeostasis in Chronic Kidney Disease: From Clinical Epidemiology to Pathophysiology.

INTRODUCTION: A simple data filtering process together with some basic concepts of control theory applied to electronically stored clinical data were used to identify some of the pathophysiological mechanisms underlying the perturbations of the calcium/phosphorus homeostasis in chronic kidney disease. MATERIAL AND METHODS: Retrospective data (a set per patient of serum single value concentrations of creatinine, calcium, phosphorus, parathormone, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D) from 2507 patients with stable chronic kidney disease not on renal replacement therapy were studied. The variables were paired and subjected sequentially to a moving average and partioned into frequency classes. The plots were interpreted using the concept of a feedback loop comprising two branches of opposite sign and of set point of the loop. The set point for each pair of variables is displaced in the course of the disease and this displacement indicates which of the two factors involved (the serum concentrations of calcium or parathormone, for example) is primarily affected. RESULTS: This analysis showed that in the course of the development of chronic kidney disease the relationships between the observed variables progressed following a monotonous, a biphasic or a triphasic pattern. DISCUSSION: As chronic kidney disease progresses, calcium/phosphorus metabolism regulation evolves through different phases. Later, there is a progressive loss of the parathyroid gland sensitivity to the control by the serum concentrations of calcium and phosphorus. The sensitivity to the inhibitory action of 1,25-dihydroxyvitamin D decreases monotonously but never releases the gland. CONCLUSION: The clinical data analysis used permits to illustrate the underlying pathophysiological mechanisms.

Introdução: O propósito do presente trabalho consistiu em descrever alguns dos mecanismos fisiopatológicos envolvidos nas perturbações do metabolismo fosfo-cálcico na doença renal crónica a partir de um processo simples de filtragem de dados e de alguns conceitos básicos de teoria de sistemas. Material e Métodos: Foram estudados os valores (um conjunto por doente, obtido numa única colheita de sangue) de creatinina, cálcio, fósforo, paratormona, 25-hidroxivitamina D e 1,25-dihidroxivitamina D num grupo de 2507 doentes com doença renal crónica não sujeitos a substituição renal. As variáveis foram emparelhadas e tratadas, primeiro por um processo de média deslizante e depois por distribuição por classes de frequência. Os resultados obtidos foram interpretados à luz do conceito de ansa de retro-controlo contendo dois ramos de sinais opostos cuja intersecção define o 'ponto operacional' do sistema. Este ponto desloca-se ao longo da evolução da doença e o sentido do deslocamento indica qual dos dois fatores (a calcémia ou a concentração de paratormona, por exemplo) está a afetar primariamente o sistema. Resultados: Esta análise mostrou que a evolução das relações entre as variáveis observadas seguiu um padrão monótono, bifásico ou trifásico. Discussão: À medida que a doença renal crónica progride ocorrem alterações na regulação do metabolismo fosfo-cálcico, passando por diferentes fases. Numa fase mais avançada há uma perda do controlo das glândulas paratiróides pela calcémia e pela fosfatémia. A sensibilidade à ação inibitória da 1,25-dihidroxivitamina D diminui progressivamente mas nunca desaparece. Conclusão: A metodologia utilizada na análise de uma base de dados clínicos permitiu ilustrar mecanismos fisiopatológicos subjacentes.

A multinodular goiter as the initial presentation of a renal cell carcinoma harbouring a novel VHL mutation.

BACKGROUND: Secondary involvement of the thyroid gland is rare. Often the origin of the tumor is difficult to identify from the material obtained by fine-needle aspiration cytology. Renal cell carcinoma of the clear-cell type is one of the more common carcinomas to metastasize to the thyroid gland. Somatic mutations of the von Hippel-Lindau tumor suppressor gene are associated with the sporadic form of this tumor. We aimed to illustrate the potential utility of DNA based technologies to search for specific molecular markers in order to establish the anatomic site of origin. CASE PRESENTATION: A 54-yr-old Caucasian male complaining of a rapidly increasing neck tumor was diagnosed as having a clear-cell tumor by fine-needle aspiration cytology. A positive staining for cytokeratin as well as for vimentin and CD10 in the absence of staining for thyroglobulin, calcitonin and TTF1 suggested a renal origin confirmed by computed tomography. Using frozen RNA, obtained from cells left inside the needle used for fine needle aspiration cytology, it was possible to identify a somatic mutation (680 delA) in the VHL gene. CONCLUSION: In the presence of a clear-cell tumor of the thyroid gland, screening for somatic mutations in the VHL gene in material derived from thyroid aspirates might provide additional information to immunocytochemical studies and therefore plays a contributory role to establish the final diagnosis. Moreover, in a near future, this piece of information might be useful to define a targeted therapy.

Expression of prolactin receptor and prolactin in normal and malignant thyroid: a tissue microarray study.

OBJECTIVE: There is increasing evidence involving prolactin (PRL) and its receptor (PRLR) in the development of different cancers. The aim of the present study was to investigate the expression of PRLR and PRL in human thyroid tissues. DESIGN AND METHODS: Using tissue microarray (TMA) by immunohistochemical staining, we examined the expression level of PRLR and PRL in 314 specimens from 71 thyroid cancer patients and 15 normal thyroid samples. RESULTS: Expression of the PRLR was observed in 93.3% of normal thyroid samples and in 76.1% of all thyroid cancers, while expression of PRL was observed in only 10% of medullary thyroid carcinomas and not at all in the other specimens, whether normal or neoplastic. Moreover, results suggested an overexpression of PRLR in 70% of medullary thyroid carcinomas, whereas 53.3% of poorly differentiated thyroid carcinomas showed a negative pattern of staining (p = 0.014 vs normal). CONCLUSIONS: Present data revealed, for the first time, a widespread expression of PRLR in normal and neoplastic human thyroid tissues as well as a scarce expression of PRL, observed only in a few medullary thyroid carcinomas. Whether the overexpression of PRLR observed in medullary thyroid carcinomas or the underexpression of PRLR observed in poorly differentiated thyroid carcinomas play a contributory role in the oncogenesis of these tumors remains to be determined.

Metastatic follicular carcinoma associated with hyperthyroidism.

PURPOSE: A 68-year-old man with metastatic follicular thyroid carcinoma had T3 hyperthyroidism. MATERIAL AND METHODS: A bone scan showed intense uptake in the thyroid and multiple areas of increased uptake in the skeleton. Hyperthyroidism persisted after total thyroidectomy. Treatment with I-131 induced a transient state of euthyroidism lasting approximately 9 months. Further tumor growth and relapse of hyperthyroidism eventually occurred and the patient died 25 months after surgery. Molecular and cytogenetic analyses were performed. RESULTS: No mutations were detected of either of the thyrotropin receptor or of the alpha subunit of the stimulatory guanine-nucleotide-binding proteins. Hyperthyroidism was unlikely the result of thyroid-stimulating receptor antibodies. Comparative genomic hybridization analysis showed that the tumor was characterized by multiple chromosomal imbalances. CONCLUSIONS: This is an unusual case of follicular thyroid carcinoma with initial high I-131 uptake by the thyroid and bone metastases and concurrent hyperthyroidism. Despite the increased I-131 uptake in the tumor, I-131 treatment only transiently controlled the hyperthyroidism and had no effect on tumor size. The cause of hyperthyroidism remained unknown. T3 predominance was unlikely the result of type 2 deiodinase overexpression because loss of genetic material was demonstrated at chromosome 14 long arm, where type 2 deiodinase is mapped.

Hyperparathyroidism-jaw tumor syndrome in Roma families from Portugal is due to a founder mutation of the HRPT2 gene.

The hyperparathyroidism-jaw tumor (HPT-JT) syndrome is an autosomal dominant disorder characterized by the occurrence of parathyroid tumors and ossifying jaw fibromas. The gene causing HPT-JT, HRPT2, is located on chromosome 1q31.2 and consists of 17 exons that encode a 531-amino acid protein, designated parafibromin. We recently identified six Roma families in Portugal with 56 members (11 affected and 45 asymptomatic), who had the HPT-JT syndrome. We postulated that they may have a common ancestor and that the HPT-JT syndrome may be due to a mutation of the HRPT2 gene. Haplotype analysis using 14 chromosome 1q24-q32 polymorphic markers showed that the 11 affected individuals shared a common haplotype defined by seven markers that spanned an approximately 12.5-cM region, flanked centromerically by D1S202 and telomerically by D1S306. DNA sequence analysis identified a 2-bp (TG or GT) frameshift deletion in exon 8, which predicts a truncated parafibromin protein, in all 11 affected individuals. This mutation was also found in 19 unaffected individuals (age range, 12-74 yr) who shared the affected haplotype, suggesting a low age-related penetrance for HPT-JT in these families. Thus, the HPT-JT syndrome in six Roma families from Portugal is due to a novel founder mutation in the HRPT2 gene.

Expression of PAX8-PPAR gamma 1 rearrangements in both follicular thyroid carcinomas and adenomas.

Recently, a translocation t(2;3)(q13;p25), leading to the formation of a chimeric PAX8-peroxisome proliferator-activated receptor (PPAR)gamma 1 oncogene, was detected in follicular thyroid carcinomas (FTC), but not in follicular thyroid adenomas (FTA), papillary thyroid carcinomas (PTC), or multinodular hyperplasias. However, previous cytogenetic studies have identified the t(2;3)(q13;p25) translocation also in some cases of FTA. In this study, we have combined RT-PCR with primers in exons 4-8 of PAX8 and in exon 1 of PPAR gamma 1 with PPAR gamma immunohistochemistry to study PAX8-PPAR gamma 1 oncogene activation in FTC (n = 9), FTA (n = 16), PTC (n = 9), anaplastic thyroid carcinomas (n = 4), and multinodular hyperplasias (n = 2). PAX8-PPAR gamma 1 rearrangements were detected by RT-PCR in 5 of 9 (56%) FTC and in 2 of 16 (13%) FTA. By contrast, all cases of PTC, anaplastic thyroid carcinomas, and multinodular hyperplasia were RT-PCR-negative. Diffuse nuclear immunoreactivity for PPAR gamma was observed in 7 of 9 (78%) FTC, 5 of 16 FTA (31%), and 1 of 9 PTC (11%). Positivity was focal in 3 cases (1 FTC, 1 PTC, and 1 multinodular hyperplasia). Diffuse nuclear staining for PPAR gamma was present in RT-PCR- negative cases of FTC (n = 3), FTA (n = 3), and PTC (n = 1), suggesting that a different PAX8-PPAR gamma 1 breakpoint, a rearrangement between PPAR gamma 1 and a non-PAX8 partner, or overexpression of the native protein might be present. Our findings that PAX8-PPAR gamma 1 rearrangements are present in both follicular carcinomas and adenomas suggest that this oncogene is not a reliable marker to differentiate between FTC and FTA in fine-needle aspiration biopsies of follicular neoplasms of the thyroid.

Immunostaining and RT-PCR: different approaches to search for RET rearrangements in patients with papillary thyroid carcinoma.

Different techniques of molecular biology have been used to screen for RET rearrangements. More recently, immunohistochemistry has been used, assuming that RET is not expressed in normal thyroid follicular cells. The present study was designed to define the prevalence of RET expression in patients with papillary thyroid carcinoma, by immunohistochemistry and by RT-PCR; to search specifically for RET/PTC-1; -2; -3 rearrangements using RT-PCR, and to compare results obtained by immunohistochemistry with those obtained by RT-PCR. Immunohistochemistry was performed using a polyclonal antibody against tyrosine kinase domain of Ret protein. Screening for RET/PTC1-3 was performed using RT-PCR and specific primers for each rearrangement; complementarily, a subset of cases were tested using RET exon 10/11 primers designed to detect the expression of the wild-type RET. Positive staining was observed in 30 of 39 (77%) tumours. RET/PTC1-3 rearrangements were detected in 8 of 32 (25%) cases. Ten of 15 (67%) cases expressed the wild-type RET. Two tumours characterised by positive immunostaining, absence of RET 5' expression and absence of RET/PTC1-3 expression were considered as expressing a RET rearrangement different from RET/PTC-1, -2, or -3. In 3 of 10 tumours, expression of the wild-type RET coexisted with the expression of a RET rearrangement. Positive staining does not necessarily mean the presence of a rearrangement; it may correspond to the expression of the wild-type RET, RET rearrangement or both. On the contrary, positive staining without evidence for the expression of the extracellular domain of RET is highly suggestive of a RET rearrangement independently of the type. Refinement of diagnosis depends on RT-PCR with specific primers.

Clonal origin of non-medullary thyroid tumours assessed by non-random X-chromosome inactivation.

OBJECTIVE: X-chromosome inactivation analysis was performed in order to assess the clonal origin of non-medullary thyroid tumours and to distinguish between multicentricity and multifocality in multiple papillary thyroid carcinoma (PTC). METHODS: One hundred and thirteen tumour samples from 31 patients with isolated PTC, 16 patients with multinodular PTC, 14 patients with follicular thyroid adenoma (FTA) and 15 patients with follicular thyroid carcinoma (FTC) were collected. The corresponding normal thyroid tissues were analysed, and in 14 cases, tumour-surrounding tissue was also studied. Genomic DNA was digested with HpaII and HhaI previous to PCR amplification of the polymorphic CAG repeat, on exon 1 of the human androgen receptor gene (HUMARA). PCR products were analysed by denaturing gel electrophoresis, silver staining and densitometric analysis. PCR products were also used to determine the number of CAG repeats of patients with isolated PTC, FTA, FTC and of 41 healthy volunteers. RESULTS: Heterozygosity for the HUMARA polymorphism was found in 64/76 (84%) cases. Lyonization of the thyroid was observed in 15/76 (20%) cases, which were excluded from clonal analysis. Except for two cases of isolated PTC, all tumour samples studied presented monoclonal X-inactivation patterns, while normal thyroid tissue was polyclonal. Monoclonal patterns were also found in 4/14 tumour-surrounding tissues. No difference was found in the length of CAG alleles between patients and controls. Of eight informative cases of multinodular PTC, three showed evidence of multicentricity and five revealed patterns consistent with multifocality. CONCLUSIONS: Both isolated and multinodular PTC as well as FTA and FTC are of monoclonal origin. Our results also suggest that approximately one-third of multiple PTC have an independent origin for the different nodules (multicentricity). Monoclonality was also found in tissues surrounding some PTC nodules. No association was found between the length of CAG alleles and thyroid malignancies.

Two novel variants in the thyroxine-binding globulin (TBG) gene behind the diagnosis of TBG deficiency.

OBJECTIVE: Search for germline mutations in the thyroxine-binding globulin (TBG) gene of two unrelated Portuguese females of Caucasian origin in whom the diagnosis of TBG deficiency was suspected because of suppressed TSH despite marginally low total thyroxine and tri-iodothyronine. DESIGN AND METHODS: Screening for germline mutations was conducted by non-radioactive PCR-SSCP analysis. The variants documented by this approach were characterized by sequencing. Moreover, in order to define whether they were mutations or polymorphisms we looked for the same variants analysing 100 alleles at random. To achieve this goal we used, alternatively, restriction analysis and the minisequencing method with an automated capillary electrophoresis system and fluorescent-labelled dideoxynucleotides. RESULTS AND CONCLUSIONS: Two novel variants, one in each patient, were identified. One, involved codon 23 (TCA-->TAA) and the other, codon 223 (CAA-->TAA). Analysis of 50 DNA samples, randomly chosen, revealed that all were homozygous for the wild variant at codon 23. One of them was heterozygous for the variant CAA-->TAA at codon 223. This sample was found to correspond to a Caucasian female in whom serum TBG proved to be not detected. Since both variants identified result in stop codons likely to induce truncated TBG proteins, they are probably responsible for the TBG phenotype observed in the individuals studied.

Molecular diagnosis of multiple endocrine neoplasia Type 2.

Multiple endocrine neoplasia Type 2 is a rare familial cancer syndrome transmitted in an autosomal dominant manner. It is characterized by the association of medullary thyroid carcinoma with pheochromocytoma and hyperparathyroidism. Medullary thyroid carcinoma, present in virtually all patients, is the principal cause of death. In 1993, germline mutations in the RET proto-oncogene were identified as the underlying cause of the syndrome. Genetic screening of at-risk family members can now be performed with high specificity and sensitivity. The ability to determine gene carrier status at a preclinical stage is of great value as it allows early prophylactic thyroidectomy. The specific RET codon mutation correlates with clinical variants of the syndrome, age at onset and aggressiveness of medullary thyroid carcinoma. This review will focus on mutational spectrum, genotype-phenotype correlations and clinical decisions based on genetic information.

The minisequencing method: a simple strategy for genetic screening of MEN 2 families.

BACKGROUND: Multiple endocrine neoplasia type 2 is an autosomal dominant disorder. MEN 2A is characterized by medullary thyroid carcinoma, pheochromocytoma and hyperparathyroidism; MEN 2B by medullary thyroid carcinoma, pheochromocytoma and characteristic stigmata. Activating germline mutations of the RET proto oncogene are responsible for this hereditary syndrome. Codon 634 mutations are the most common mutations occurring in MEN 2A families whereas a specific mutation at codon 918 is observed in the great majority of MEN 2B families. Analysis of these codons will provide a final diagnosis in the great majority of affected families making unnecessary further studies. To specifically study the codons 634 and 918 we used a minisequencing method as an alternative method to complete sequencing. RESULTS: Using this mutation detection method we were able to reproduce in all cases, representative of 7 families, the information previously obtained by direct sequencing of PCR products. Depending on the number of primers used in the minisequencing reaction, we were able to interrogate either only one nucleotide of the target codon or the three nucleotides simultaneously. CONCLUSIONS: This technique appears as a simple, rapid and efficient method for genetic screening of MEN 2 families. It can be utilized to seek for unknown mutations at specific codons or to screen for previously identified mutations and is therefore of interest to study index cases or individuals at risk. Results suggest that complete sequencing is unnecessary.

Preoperative diagnosis of suspicious parathyroid adenomas by RT-PCR using mRNA extracted from leftover cells in a needle used for ultrasonically guided fine needle aspiration cytology.

OBJECTIVE: To determine the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) detection of parathormone (PTH) gene mRNA in needle aspirates to confirm the parathyroid nature of suspicious cervical lesions in patients with hyperparathyroidism. STUDY DESIGN: Ultrasound-guided fine needle aspiration cytology (FNAC) was performed on 12 patients with suspected parathyroid adenomas. The aspirates were subjected to cytologic and chromogranin A examination. Messenger RNA was isolated from left-over cells within the needle, and RT-PCR was used to amplify mRNA encoding PTH. RESULTS: The 12 aspirates were positive by PTH RT-PCR, and molecular diagnosis was confirmed by histologic examination. The lower sensitivity of cytologic and immunocytochemical methods was demonstrated, respectively, by 67% and 50% of positive results. Sensitivity assays demonstrated that with our system, PTH RT-PCR products are obtained from as few as 10 pg of parathyroid mRNA. CONCLUSION: The present study showed that RT-PCR-based analysis of PTH gene transcripts in aspirates, obtained by US-guided FNAC of cervical lesions suspicious for parathyroid adenoma, is a feasible, sensitive and specific method for preoperative diagnosis of parathyroid adenomas.

Identification of de novo germline mutations in the HRPT2 gene in two apparently sporadic cases with challenging parathyroid tumor diagnoses.

The diagnosis of parathyroid carcinomas is often difficult. HRPT2 mutations have been identified in familial [hyperparathyroidism-jaw tumor (HPT-JT) syndrome] and sporadic parathyroid carcinomas, supporting that HRPT2 mutations may confer a malignant potential to parathyroid tumors. In this study, we report the clinical, histopathological, and genetic investigation of two unrelated cases, whom had apparently sporadic malignant parathyroid tumors, initially diagnosed as adenomas. In one case, the differential diagnosis was complicated by cervical seeding of parathyroid tumor cells. Genetic studies identified de novo HRPT2 germline mutations in cases 1 (c.518_521delTGTC [p.Ser174LysfsX27]) and 2 (c.226 C > T [p.Arg76X]), unveiling the hereditary HPT-JT syndrome in both patients. Furthermore, the identification of somatic mutations in the patients? parathyroid tumors provided evidence for complete inactivation of the HRPT2 gene, which was consistent with the tumor malignant features. The sensitivity of parafibromin immunostaining to detect HRPT2 mutations was limited. The present data suggests that patients with apparently sporadic parathyroid carcinomas, or parathyroid tumors with atypical histological features, should undergo molecular genetic testing, as it may detect germline HRPT2 mutations. Establishing the diagnosis of hereditary HPT-JT syndrome is relevant for clinical counseling and management of the carriers and their relatives.

Clinical and genetic characterization of Portuguese patients with pseudohypoparathyroidism type Ib.

Patients with pseudohypoparathyroidism type Ib (PHP-Ib) present hypocalcemia and hyperphosphatemia, as a consequence of a resistance to PTH action, through its G-protein-coupled receptor, in the renal tubules. This resistance results from tissue-specific silencing of the G-protein alpha-subunit (G(s)α), due to imprinting disruption of its encoding locus--GNAS. In familial PHP-Ib, maternally inherited deletions at the STX16 gene are associated to a regional GNAS methylation defect. In sporadic PHP-Ib, broad methylation changes at GNAS arise from unknown genetic causes. In this study, we describe the clinical presentation of PHP-Ib in four Portuguese patients (two of whom were siblings), and provide further insight for the management of patients with this disease. The diagnosis of PHP-Ib was made after detection of GNAS imprinting defects in each of the cases. In the siblings, a regional GNAS methylation change resulted from a known 3.0 kb STX16 deletion. In the other two patients, the broad methylation defects at GNAS, which were absent in their relatives, resulted from genetic alterations that remain to be identified. We report the first clinical and genetic study of Portuguese patients with PHP-Ib. The genetic identification of a hereditary form of this rare disease allowed an early diagnosis, and may prevent hypocalcemia-related complications.