Whole-exome identifies RXRG and TH germline variants in familial isolated prolactinoma.

Familial isolated pituitary adenoma (FIPA) is a rare genetic disorder. In a subset of FIPA families AIP germline mutations have been reported, but in most FIPA cases the exact genetic defect remains unknown. The present study aimed to determine the genetic basis of FIPA in a Brazilian family. Three siblings presented with isolated prolactin genes. Further mutation screening was performed using whole-exome sequencing and all likely causative mutations were validated by Sanger sequencing. In silico analysis and secreting pituitary adenoma diagnosed through clinical, biochemical and imaging testing. Sanger sequencing was used to genotype candidate prolactinoma-mutated additional predictive algorithms were applied to prioritize likely pathogenic variants. No mutations in the coding and flanking intronic regions in the MEN1, AIP and PRLR genes were detected. Whole-exome sequencing of three affected siblings revealed novel, predicted damaging, heterozygous variants in three different genes: RXRG, REXO4 and TH. In conclusion, the RXRG and TH possibly pathogenic variants may be associated with isolated prolactinoma in the studied family. The possible contribution of these genes to additional FIPA families should be explored.

HRPT2-related familial isolated hyperparathyroidism: could molecular studies direct the surgical approach? / Formas familiares de hiperparatireoidismo relacionadas a mutações no gene HRPT2: estudos moleculares poderiam direcionar procedimentos cirúrgicos?

It is still debatable which is the best management to familial forms of hyperparathyroidism. Conservative, minimally invasive or aggressive surgical approaches have been proposed from different groups around the world. Our objective was to study the gene mutation, expression of HRPT2 and the clinical outcome after 32 years of follow-up in one Brazilian kindred with familial isolated hyperparathyroidism (FIHP). Clinical and biochemical data, direct sequencing of the HRPT2 gene, analysis of parafibromin expression using RT-PCR, and immunohistochemistry were done. A nonsense mutation was found in exon 1 (c.96G>A)(p.Trp32X) in all affected members studied. Using RT-PCR, mRNA transcription was altered with complete absence of both transcripts in tumor tissue. Immunohistochemical analysis of tumors showed loss of parafibromin immunoreactivity. In this kindred there was a high prevalence of recurrence (75 percent), or persistence after less than subtotal parathyroidectomy that led us to consider a more aggressive surgical approach should be discussed among the affected family members, once surgical criteria was met. We concluded that it is necessary to individualize the surgical approach for HRPT2-related hyperparathyroidism until we can gather a better phenotype-genotype correlation in larger series, to best define their treatment.

A melhor conduta nas formas familiares de hiperparatireoidismo relacionadas a mutações no gene HRPT2 ainda é controvertida. Cirurgias conservadoras, minimamente invasivas ou mais agressivas já foram propostas por diferentes grupos. Objetivamos estudar a seqüência e a expressão do gene HRPT2, além do desfecho clínico, após seguimento de até 32 anos de uma família brasileira com hiperparatireodismo familiar isolado (FIHP). Utilizamos dados clínicos e bioquímicos, seqüenciamento direto do HRPT2 além de análise da expressão da parafibromina através da RT-PCR e imunohistoquímica. Foi identificada mutação nonsense no éxon 1 (c.96G>A)(p.Trp32X) em todos os membros afetados que foram estudados. A análise do mRNA transcrito, através da RT-PCR, demonstrou ausência do transcrito no tecido tumoral. A imunohistoquímica também evidenciou ausência da parafibromina. Nessa família houve alta (75 por cento) prevalência de recorrência ou persistência da doença após paratireoidectomia parcial o que nos levou a considerar fundamental discutir uma abordagem cirúrgica mais agressiva com os outros familiares portadores da mutação caso critérios de indicação cirúrgica sejam atingidos. Dessa maneira, até que estudos mais amplos estabeleçam uma correlação genótipo-fenótipo no hiperparatireoidismo familiar relacionado a mutações no HRPT2, a abordagem cirúrgica deverá ser individualizada.

Familial hyperparathyroidism: surgical outcome after 30 years of follow-up in three families with germline HRPT2 mutations.

BACKGROUND: Familial forms of hyperparathyroidism are responsible for approximately 10% of the cases of primary hyperparathyroidism, and their management is different from the sporadic forms. Our objective was to study the gene sequence and expression of HRPT2 and clinical outcome regarding recurrence or persistence rates in three Brazilian kindreds with familial hyperparathyroidism after up to 30 years of follow-up. METHODS: Clinical and biochemical data, direct sequencing of germline DNA of the HRPT2 gene, and analysis of parafibromin expression (HRPT2 gene product) using RT-PCR and immunohistochemistry of resected parathyroid neoplasms were performed. RESULTS: Affected members of kindred A were found to carry a novel, germline, nonsense mutation in exon 1 (c.96G>A; W32X) of HRPT2. Six of seven patients who have undergone less than total parathyroidectomy recurred after up to 30 years of follow-up. An unrelated affected patient from kindred B had a germline mutation in exon 7 (c.686delGAGT), and the disease recurred with several pulmonary metastases after 5 years follow-up. The affected member of kindred C also had a previously described mutation in exon 7 (c.679delAG) and the disease recurred after 10 years of follow-up. All parathyroid neoplasms from these families had diffuse loss of expression by immunohistochemistry. CONCLUSIONS: An unacceptable recurrence/persistence rate (80%) associated with increasingly difficult re-operations and risk of parathyroid carcinoma in the setting of germline mutations of HRPT2 gene with familial hyperparathyroidism suggest that a more aggressive operative approach should be undertaken in these patients. Parafibromin immunohistochemistry may serve as a cost-effective screen for HRPT2-related aggressive parathyroid disease.

HRPT2-related familial isolated hyperparathyroidism: could molecular studies direct the surgical approach?

It is still debatable which is the best management to familial forms of hyperparathyroidism. Conservative, minimally invasive or aggressive surgical approaches have been proposed from different groups around the world. Our objective was to study the gene mutation, expression of HRPT2 and the clinical outcome after 32 years of follow-up in one Brazilian kindred with familial isolated hyperparathyroidism (FIHP). Clinical and biochemical data, direct sequencing of the HRPT2 gene, analysis of parafibromin expression using RT-PCR, and immunohistochemistry were done. A nonsense mutation was found in exon 1 (c.96G>A)(p.Trp32X) in all affected members studied. Using RT-PCR, mRNA transcription was altered with complete absence of both transcripts in tumor tissue. Immunohistochemical analysis of tumors showed loss of parafibromin immunoreactivity. In this kindred there was a high prevalence of recurrence (75%), or persistence after less than subtotal parathyroidectomy that led us to consider a more aggressive surgical approach should be discussed among the affected family members, once surgical criteria was met. We concluded that it is necessary to individualize the surgical approach for HRPT2-related hyperparathyroidism until we can gather a better phenotype-genotype correlation in larger series, to best define their treatment.

Association between decreased WWOX protein expression and thyroid cancer development.

CONTEXT: Chromosomal fragile sites are often related to cancer development. The WW domain-containing oxidoreductase gene (WWOX) spans the second most common chromosomal fragile site (FRA16D) and encodes an important proapoptotic protein. OBJECTIVE: To verify our hypothesis that underexpression of WWOX could contribute to malignant transformation of the thyroid cells. METHOD: We compared WWOX expression among follicular adenomas (FAs) and differentiated thyroid carcinomas [follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas (PTCs)] in 53 thyroid tumors resected from patients submitted to total thyroidectomy. DESIGN: Multiple fields of tumor areas of FAs, FTCs, and PTCs as well as normal thyroid tissue were stained with WWOX antiserum, and classified by the extent of staining (percentage of cells staining) and staining intensity. MAIN OUTCOME: PTCs showed a significantly decreased expression of WWOX when compared to FAs and FTCs. Further, using a unique model of comparison in patients in whom FAs and PTCs were concomitantly present, we detected the same result (i.e., no expression in PTCs). CONCLUSION: We conclude that WWOX underexpression is an important step that might increase the vulnerability to the carcinogenesis process in PTCs.

Large germline deletions of mitochondrial complex II subunits SDHB and SDHD in hereditary paraganglioma.

More than 30% of adrenal pheochromocytomas are hereditary. These neuroendocrine tumors are major components of three inherited cancer syndromes: multiple endocrine neoplasia type 2, von Hippel-Lindau disease (VHL), and pheochromocytoma/paraganglioma syndrome (PC/PGL). Germline mutations in RET; VHL; and SDHB, SDHC, and SDHD are associated with multiple endocrine neoplasia type 2, VHL, and PC/PGL, respectively. The majority (>70%) of hereditary extraadrenal PCs [catecholamine-secreting paragangliomas (PGL)] are accounted for by germline intragenic mutations in SDHB, SDHC, or SDHD. Therefore, a subset of hereditary PGL is not accounted for. Here we report two unrelated hereditary PGL families, one with a germline whole-gene deletion of SDHD (family 4194), the other a partial deletion of SDHB (family BRZ01). Although they were initially designated mutation negative for all of the PC-associated genes after PCR-based analysis, we suspected that a large deletion or rearrangement might be present. Genotyping around the PC-associated genes demonstrated that both families were consistent with linkage with one of these genes. Using fine structure genotyping and semiquantitative duplex PCR analysis, we identified an approximately 96-kb deletion spanning SDHD in family 4194 and an approximately 1-kb deletion involving the 5' end of SDHB in family BRZ01. Thus, including SDHB and SDHD deletion analysis could increase gene-testing sensitivity for PGL patients, which would aid in genetic counseling and management of patients and families.

Two intronic mutations cause 17-hydroxylase deficiency by disrupting splice acceptor sites: direct demonstration of aberrant splicing and absent enzyme activity by expression of the entire CYP17 gene in HEK-293 cells.

To date, only two among 46 mutations in the CYP17 gene cause 17-hydroxylase deficiency (17OHD) by disrupting mRNA splice donor sites. We studied two subjects with intronic CYP17 mutations: a compound heterozygote for Y329D plus an AG to CG substitution at the 3' end of intron 2, and a homozygote for a TTTT deletion near the 3' end of intron 3. We hypothesized that both mutations caused 17OHD by disrupting splice acceptor sites. To prove this mechanism, the entire CYP17 genes (wild type and both mutations) were amplified, subcloned into pcDNA3, and expressed in HEK-293 cells. The mRNA derived from the wild-type CYP17 gene was correctly spliced and translated into active enzyme, as shown by the correct sequence in the RT-PCR products and by the 17-hydroxylation of progesterone. In contrast, cells expressing the mutant genes had no 17-hydroxylase activity. The mRNA derived from the AG to CG mutation used the first AG in exon 3 as the splice acceptor site, shifting the reading frame and introducing a stop codon. RNA derived from the TTTT deletion skipped exon 4 entirely, deleting 29 amino acids in-frame. Our data show that these are the first two 17OHD cases resulting from mutations that alter splice acceptor sites. These studies also demonstrate the feasibility of expressing the entire CYP17 gene, with simultaneous protein and RNA analysis, as a general methodology for characterizing how intronic CYP17 mutations cause 17OHD.

Mecanismos moleculares de açäo hormonal / Molecular mechanisms of hormonal action

A característica principal dos hormônios é a sua habilidade em interagir com receptores altamente seletivos e ativar vias intracelulares do sinalizaçäo nos órgaos específicos. Após a interaçäo dos hormônios com seus receptores, uma seqüência de reaçöes pode levar ao aumento ou diminuiçäo na atividade de determinadas enzimas que, por sua vez, produzem a resposta fisiológica. Os hormônios säo bioquimicamente classificados em esteróides, peptídeos ou aminas e seus receptores diferem, basicamente, por sua localizaçäo, intra ou extracelular. No presente trabalho, o mecanismo molecular de açäo dos hormônios peptídicos (hidrofílicos) e esteróides (lipofílicos) é discutido.

Mecanismos moleculares dos tumores endócrinos / Molecular mechanisms of the endocrine tumors

Uma característica fundamental de todas as células eucariotas superiores é o período definido de vida do organismo, propriedade essa que se estende às células somáticas individuais que possuem o crescimento e a divisao altamente regulados. O estudo dos mecanismos de oncogênese tem permitido um conhecimento mais aprofundado desses processos de diferenciaçao e proliferaçao celulares. Alteraçoes genéticas relacionadas aos oncogenes, genes supressores de tumor e genes de reparo de erros de pareamento do DNA, estao ligadas à origem da formaçao dos tumores. O objetivo desta revisao é apresentar, sumariamente, alguns dos principais mecanismos de oncogênese nos tumores endócrinos.