RESUMO
Most aldosterone-producing adenomas (APAs) have gain-of-function somatic mutations of ion channels or transporters. However, their frequency in aldosterone-producing cell clusters of normal adrenal gland suggests a requirement for codriver mutations in APAs. Here we identified gain-of-function mutations in both CTNNB1 and GNA11 by whole-exome sequencing of 3/41 APAs. Further sequencing of known CTNNB1-mutant APAs led to a total of 16 of 27 (59%) with a somatic p.Gln209His, p.Gln209Pro or p.Gln209Leu mutation of GNA11 or GNAQ. Solitary GNA11 mutations were found in hyperplastic zona glomerulosa adjacent to double-mutant APAs. Nine of ten patients in our UK/Irish cohort presented in puberty, pregnancy or menopause. Among multiple transcripts upregulated more than tenfold in double-mutant APAs was LHCGR, the receptor for luteinizing or pregnancy hormone (human chorionic gonadotropin). Transfections of adrenocortical cells demonstrated additive effects of GNA11 and CTNNB1 mutations on aldosterone secretion and expression of genes upregulated in double-mutant APAs. In adrenal cortex, GNA11/Q mutations appear clinically silent without a codriver mutation of CTNNB1.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Subunidades alfa de Proteínas de Ligação ao GTP/genética , beta Catenina/genética , Adolescente , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/patologia , Adulto , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Hiperaldosteronismo/patologia , Masculino , Menopausa/metabolismo , Pessoa de Meia-Idade , Gravidez , Puberdade/metabolismoRESUMO
Aldosterone producing adenomas (APAs) occur in the adrenal glands of around 30% of patients with primary aldosteronism, the most common form of secondary hypertension. Somatic mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D and CTNNB1 have been described in ~60% of these tumours. We subjected 15 aldosterone producing adenomas (13 with known mutations and two without) to RNA Sequencing and Whole Genome Sequencing (n = 2). All known mutations were detected in the RNA-Seq reads, and mutations in ATP2B3 (G123R) and CACNA1D (S410L) were discovered in the tumours without known mutations. Adenomas with CTNNB1 mutations showed a large number of differentially expressed genes (1360 compared to 106 and 75 for KCNJ5 and ATP1A1/ATP2B3 respectively) and clustered together in a hierarchical clustering analysis. RT-PCR in an extended cohort of 49 APAs confirmed higher expression of AFF3 and ISM1 in APAs with CTNNB1 mutations. Investigation of the expression of genes involved in proliferation and apoptosis revealed subtle differences between tumours with and without CTNNB1 mutations. Together our results consolidate the notion that CTNNB1 mutations characterize a distinct subgroup of APAs.
Assuntos
Adenoma/genética , Adenoma Adrenocortical/genética , Aldosterona/genética , Transcriptoma/genética , beta Catenina/genética , Adenoma/patologia , Glândulas Suprarrenais/patologia , Adenoma Adrenocortical/patologia , Adulto , Idoso , Aldosterona/biossíntese , Canais de Cálcio Tipo L/genética , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Hiperaldosteronismo/genética , Hiperaldosteronismo/patologia , Masculino , Pessoa de Meia-Idade , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Análise de Sequência de RNA , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Aldosterone-producing adenomas (APA) are more common than initially anticipated. APA cause primary aldosteronism (PA), which affect 3-10% of the hypertensive population. Research during recent years has led to an increased knowledge of the background dysregulation of the increased aldosterone release, where mutation in the gene encoding the potassium channel GIRK4-KCNJ5-is the most common. Moreover, the discovery of aldosterone-producing cell clusters in apparently normal adenomas has also led to increased understanding of the development of PA, and presumably also APA. A continuum ranging from low-renin hypertension to APA and overt PA is reasoned, and the secondary effects of aldosterone on especially the cardiovascular system have also become more evident. Diagnostics of PA and APA is important in order to reduce cardiovascular morbidity and mortality, but the diagnostic methods are somewhat unspecific and insensitive, indicating the need for novel methods.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Aldosterona/metabolismo , Hiperaldosteronismo/etiologia , Neoplasias do Córtex Suprarrenal/patologia , Neoplasias do Córtex Suprarrenal/terapia , Adenoma Adrenocortical/patologia , Adenoma Adrenocortical/terapia , Predisposição Genética para Doença , Humanos , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Hiperaldosteronismo/terapia , MutaçãoRESUMO
The Wnt/ß-Catenin signaling pathway is one of the most frequently altered pathways in adrenocortical carcinomas (ACCs). The aim of this study was to investigate the status of Wnt/ß-Catenin signaling pathway by analyzing the expression level of ß-Catenin and the mutational status of APC, AXIN2, CTNNB1, and ZNRF3 in ACCs. Mutations in APC, CTNNB1, ZNRF3 and homozygous deletions in ZNRF3 were observed in 3.8% (2/52), 11.5% (6/52), 1.9% (1/52) and 17.3% (9/52) of the cohort respectively. Novel interstitial deletions in CTNNB1 spanning intron 1 to exon 3/intron 3 were also found in 7.7% (4/52) of the tumours. All the observed alterations were mutually exclusive. Nuclear accumulation of ß-Catenin, increased expression of Cyclin D1 and significantly higher expression of AXIN2 (p = 0.0039), ZNRF3 (p = 0.0032) and LEF1(p = 0.0090) observed in the tumours harbouring the deletion in comparison to tumours without CTNNB1 mutation demonstrates that the truncated ß-Catenin is functionally active and erroneously activates the downstream targets. Significantly lower overall survival rate in patients with tumours harbouring alterations in APC/CTNNB1/ZNRF3 in comparison to those without mutation was observed. In conclusion, the discovery of novel large deletions in addition to the point mutations in CTNNB1 infers that activation of Wnt/ß-Catenin pathway via alterations in CTNNB1 occurs frequently in ACCs. We also confirm that alterations in Wnt/ß-Catenin signaling pathway members have a negative effect on overall survival of patients.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/genética , Carcinoma Adrenocortical/patologia , Mutação Puntual , Deleção de Sequência , beta Catenina/genética , Adulto , Idoso , Núcleo Celular/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , beta Catenina/análiseRESUMO
Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors arising in the medullae of the adrenal glands or in paraganglia. The knowledge of the tumor biology of these lesions has increased dramatically during the past two decades and more than a dozen recurrently mutated genes have been identified. Different clusters have been described that share epigenetic signatures. Mutations in the succinate dehydrogenase complex subunit genes play a pivotal role in reprogramming the epigenetic state of these tumors by inhibiting epigenetic regulators such as TET enzymes and histone demethylases. Another subgroup of tumors carries hypomethylated genomes, and overexpression of several micro-RNAs has been described. While much remains to be investigated regarding the epigenetics of PPGLs, it is clear that it plays an important role in PPGL biology.
Assuntos
Epigênese Genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais , Animais , Metilação de DNA/genética , Impressão Genômica , Humanos , Paraganglioma , Feocromocitoma/patologiaRESUMO
The genetics behind predisposition to small intestinal neuroendocrine tumors (SI-NETs) is largely unknown, but there is growing awareness of a familial form of the disease. We aimed to identify germline mutations involved in the carcinogenesis of SI-NETs. The strategy included next-generation sequencing of exome- and/or whole-genome of blood DNA, and in selected cases, tumor DNA, from 24 patients from 15 families with the history of SI-NETs. We identified seven candidate mutations in six genes that were further studied using 215 sporadic SI-NET patients. The result was compared with the frequency of the candidate mutations in three control cohorts with a total of 35,688 subjects. A heterozygous variant causing an amino acid substitution p.(Gly396Asp) in the MutY DNA glycosylase gene (MUTYH) was significantly enriched in SI-NET patients (minor allele frequencies 0.013 and 0.003 for patients and controls respectively) and resulted in odds ratio of 5.09 (95% confidence interval 1.56-14.74; P value = 0.0038). We also found a statistically significant difference in age at diagnosis between familial and sporadic SI-NETs. MUTYH is involved in the protection of DNA from mutations caused by oxidative stress. The inactivation of this gene leads to specific increase of G:C- > T:A transversions in DNA sequence and has been shown to cause various cancers in humans and experimental animals. Our results suggest that p.(Gly396Asp) in MUTYH, and potentially other mutations in additional members of the same DNA excision-repair pathway (such as the OGG1 gene) might be involved in driving the tumorigenesis leading to familial and sporadic SI-NETs.
Assuntos
DNA Glicosilases/genética , Neoplasias Intestinais/genética , Tumores Neuroendócrinos/genética , Adulto , Idoso , DNA Glicosilases/metabolismo , Feminino , Mutação em Linhagem Germinativa , Humanos , Neoplasias Intestinais/metabolismo , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/metabolismoRESUMO
Autoimmune polyendocrine syndrome type 1 (APS1) is a rare monogenic autoimmune disorder caused by mutations in the autoimmune regulator (AIRE) gene. High titer autoantibodies are a characteristic feature of APS1 and are often associated with particular disease manifestations. Pituitary deficits are reported in up to 7% of all APS1 patients, with immunoreactivity to pituitary tissue frequently reported. We aimed to isolate and identify specific pituitary autoantigens in patients with APS1. Immunoscreening of a pituitary cDNA expression library identified endothelin-converting enzyme (ECE)-2 as a potential candidate autoantigen. Immunoreactivity against ECE-2 was detected in 46% APS1 patient sera, with no immunoreactivity detectable in patients with other autoimmune disorders or healthy controls. Quantitative-PCR showed ECE-2 mRNA to be most abundantly expressed in the pancreas with high levels also in the pituitary and brain. In the pancreas ECE-2 was co-expressed with insulin or somatostatin, but not glucagon and was widely expressed in GH producing cells in the guinea pig pituitary. The correlation between immunoreactivity against ECE-2 and the major recognized clinical phenotypes of APS1 including hypopituitarism was not apparent. Our results identify ECE-2 as a specific autoantigen in APS1 with a restricted neuroendocrine distribution.
Assuntos
Autoantígenos/imunologia , Enzimas Conversoras de Endotelina/imunologia , Poliendocrinopatias Autoimunes/imunologia , Adolescente , Processamento Alternativo , Autoanticorpos/imunologia , Autoantígenos/genética , Autoimunidade , Criança , Enzimas Conversoras de Endotelina/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Loci Gênicos , Humanos , Imuno-Histoquímica , Masculino , Fenótipo , Hipófise/imunologia , Hipófise/metabolismo , Poliendocrinopatias Autoimunes/diagnóstico , Poliendocrinopatias Autoimunes/genéticaRESUMO
Pheochromocytomas and paragangliomas (PPGLs) are rare and frequently heritable neural-crest derived tumours arising from the adrenal medulla or extra-adrenal chromaffin cells respectively. The majority of PPGL tumours are benign and do not recur with distant metastases. However, a sizeable fraction of these tumours secrete vasoactive catecholamines into the circulation causing a variety of symptoms including hypertension, palpitations and diaphoresis. The genetic landscape of PPGL has been well characterized and more than a dozen genes have been described as recurrently mutated. Recent studies of DNA-methylation have revealed distinct clusters of PPGL that share DNA methylation patterns and driver mutations, as well as identified potential biomarkers for malignancy. However, these findings have not been adequately validated in independent cohorts. In this study we use an array-based genome-wide approach to study the methylome of 39 PPGL and 4 normal adrenal medullae. We identified two distinct clusters of tumours characterized by different methylation patterns and different driver mutations. Moreover, we identify genes that are differentially methylated between tumour subcategories, and between tumours and normal tissue.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Metilação de DNA , Epigênese Genética , Epigenômica , Feocromocitoma/genética , Adolescente , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Variações do Número de Cópias de DNA , Epigenômica/métodos , Feminino , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Masculino , Pessoa de Meia-Idade , Feocromocitoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Adulto JovemRESUMO
Primary aldosteronism (PA) is the most common cause of secondary hypertension with a prevalence of 5-10% in unreferred hypertensive patients. Aldosterone producing adenomas (APAs) constitute a large proportion of PA cases and represent a surgically correctable form of the disease. The WNT signaling pathway is activated in APAs. In other tumors, a frequent cause of aberrant WNT signaling is mutation in the CTNNB1 gene coding for ß-catenin. Our objective was to screen for CTNNB1 mutations in a well-characterized cohort of 198 APAs. Somatic CTNNB1 mutations were detected in 5.1% of the tumors, occurring mutually exclusive from mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. All of the observed mutations altered serine/threonine residues in the GSK3ß binding domain in exon 3. The mutations were associated with stabilized ß-catenin and increased AXIN2 expression, suggesting activation of WNT signaling. By CYP11B2 mRNA expression, CYP11B2 protein expression, and direct measurement of aldosterone in tumor tissue, we confirmed the ability for aldosterone production. This report provides compelling evidence that aberrant WNT signaling caused by mutations in CTNNB1 occur in APAs. This also suggests that other mechanisms that constitutively activate the WNT pathway may be important in APA formation.
Assuntos
Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Aldosterona/biossíntese , Mutação , Proteínas de Neoplasias , Via de Sinalização Wnt/genética , beta Catenina , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Aldosterona/genética , Éxons , Feminino , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Domínios Proteicos , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of ß cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/ß cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R(-/-) islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.
Assuntos
Endocanabinoides/metabolismo , Ilhotas Pancreáticas/embriologia , Morfogênese/fisiologia , Receptor CB1 de Canabinoide/metabolismo , Canais de Cátion TRPV/metabolismo , Análise de Variância , Animais , Ácidos Graxos Ômega-3/administração & dosagem , Feminino , Feto/metabolismo , Teste de Tolerância a Glucose , Processamento de Imagem Assistida por Computador , Ilhotas Pancreáticas/anatomia & histologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , GravidezRESUMO
BACKGROUND: Recent studies have demonstrated equal quality of targeted next generation sequencing (NGS) compared to Sanger Sequencing. Whereas these novel sequencing processes have a validated robust performance, choice of enrichment method and different available bioinformatic software as reliable analysis tool needs to be further investigated in a diagnostic setting. METHODS: DNA from 21 patients with genetic variants in SDHB, VHL, EPAS1, RET, (n=17) or clinical criteria of NF1 syndrome (n=4) were included. Targeted NGS was performed using Truseq custom amplicon enrichment sequenced on an Illumina MiSEQ instrument. Results were analysed in parallel using three different bioinformatics pipelines; (1) Commercially available MiSEQ Reporter, fully automatized and integrated software, (2) CLC Genomics Workbench, graphical interface based software, also commercially available, and ICP (3) an in-house scripted custom bioinformatic tool. RESULTS: A tenfold read coverage was achieved in between 95-98% of targeted bases. All workflows had alignment of reads to SDHA and NF1 pseudogenes. Compared to Sanger sequencing, variant calling revealed a sensitivity ranging from 83 to 100% and a specificity of 99.9-100%. Only MiSEQ reporter identified all pathogenic variants in both sequencing runs. CONCLUSIONS: We conclude that targeted next generation sequencing have equal quality compared to Sanger sequencing. Enrichment specificity and the bioinformatic performance need to be carefully assessed in a diagnostic setting. As acceptable accuracy was noted for a fully automated bioinformatic workflow, we suggest that processing of NGS data could be performed without expert bioinformatics skills utilizing already existing commercially available bioinformatics tools.
Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Feocromocitoma/diagnóstico , Adolescente , Neoplasias das Glândulas Suprarrenais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Feocromocitoma/genética , Estudos Retrospectivos , Adulto JovemRESUMO
Aldosterone-producing adenomas (APAs) are found in 1.5-3.0% of hypertensive patients in primary care and can be cured by surgery. Elucidation of genetic events may improve our understanding of these tumors and ultimately improve patient care. Approximately 40% of APAs harbor a missense mutation in the KCNJ5 gene. More recently, somatic mutations in CACNA1D, ATP1A1 and ATP2B3, also important for membrane potential/intracellular Ca(2) (+) regulation, were observed in APAs. In this study, we analyzed 165 APAs for mutations in selected regions of these genes. We then correlated mutational findings with clinical and molecular phenotype using transcriptome analysis, immunohistochemistry and semiquantitative PCR. Somatic mutations in CACNA1D in 3.0% (one novel mutation), ATP1A1 in 6.1% (six novel mutations) and ATP2B3 in 3.0% (two novel mutations) were detected. All observed mutations were located in previously described hotspot regions. Patients with tumors harboring mutations in CACNA1D, ATP1A1 and ATP2B3 were operated at an older age, were more often male and had tumors that were smaller than those in patients with KCNJ5 mutated tumors. Microarray transcriptome analysis segregated KCNJ5 mutated tumors from ATP1A1/ATP2B3 mutated tumors and those without mutation. We observed significant transcription upregulation of CYP11B2, as well as the previously described glomerulosa-specific gene NPNT, in ATP1A1/ATP2B3 mutated tumors compared to KCNJ5 mutated tumors. In summary, we describe novel somatic mutations in proteins regulating the membrane potential/intracellular Ca(2) (+) levels, and also a distinct mRNA and clinical signature, dependent on genetic alteration.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/metabolismo , Biomarcadores Tumorais/genética , Hiperaldosteronismo/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Canais de Cálcio Tipo L/genética , Feminino , Seguimentos , Perfilação da Expressão Gênica , Humanos , Hiperaldosteronismo/metabolismo , Hiperaldosteronismo/patologia , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Reação em Cadeia da Polimerase , Prognóstico , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
PURPOSE: Pheochromocytoma and paraganglioma (PPGL) patients display heterogeneity in the clinical presentation and underlying genetic cause. The degree of inter- and intratumor genetic heterogeneity has not yet been defined. EXPERIMENTAL DESIGN: In PPGLs from 94 patients, we analyzed LOH, copy-number variations, and mutation status of SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, NF1, RET, TMEM127, MAX, and HRAS using high-density SNP array and targeted deep sequencing, respectively. Genetic heterogeneity was determined through (i) bioinformatics analysis of individual samples that estimated absolute purity and ploidy from SNP array data and (ii) comparison of paired tumor samples that allowed reconstruction of phylogenetic trees. RESULTS: Mutations were found in 61% of the tumors and correlated with specific patterns of somatic copy-number aberrations (SCNA) and degree of nontumoral cell admixture. Intratumor genetic heterogeneity was observed in 74 of 136 samples using absolute bioinformatics estimations and in 22 of 24 patients by comparison of paired samples. In addition, a low genetic concordance was observed between paired primary tumors and distant metastases. This allowed for reconstructing the life history of individual tumors, identifying somatic mutations as well as copy-number loss of 3p and 11p (VHL subgroup), 1p (Cluster 2), and 17q (NF1 subgroup) as early events in PPGL tumorigenesis. CONCLUSIONS: Genomic landscapes of PPGL are specific to mutation subtype and characterized by genetic heterogeneity both within and between tumor lesions of the same patient.
Assuntos
Transformação Celular Neoplásica/genética , Heterogeneidade Genética , Feocromocitoma/genética , Feocromocitoma/patologia , Evolução Clonal/genética , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Instabilidade Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Metástase Neoplásica , Estadiamento de Neoplasias , Filogenia , Ploidias , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Insulinomas are pancreatic islet tumors that inappropriately secrete insulin, producing hypoglycemia. Exome and targeted sequencing revealed that 14 of 43 insulinomas harbored the identical somatic mutation in the DNA-binding zinc finger of the transcription factor Yin Yang 1 (YY1). Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that this T372R substitution changes the DNA motif bound by YY1. Global analysis of gene expression demonstrated distinct clustering of tumors with and without YY1(T372R) mutations. Genes showing large increases in expression in YY1(T372R) tumors included ADCY1 (an adenylyl cyclase) and CACNA2D2 (a Ca(2+) channel); both are expressed at very low levels in normal ß-cells and show mutation-specific YY1 binding sites. Both gene products are involved in key pathways regulating insulin secretion. Expression of these genes in rat INS-1 cells demonstrated markedly increased insulin secretion. These findings indicate that YY1(T372R) mutations are neomorphic, resulting in constitutive activation of cAMP and Ca(2+) signaling pathways involved in insulin secretion.
Assuntos
Regulação da Expressão Gênica , Insulinoma/genética , Mutação de Sentido Incorreto , Neoplasias Pancreáticas/genética , Fator de Transcrição YY1/genética , Adenilil Ciclases/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Sítios de Ligação , Glicemia/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Estudos de Coortes , AMP Cíclico/metabolismo , Feminino , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Insulinoma/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neoplasias Pancreáticas/metabolismo , Ligação Proteica , Fator de Transcrição YY1/metabolismoRESUMO
BACKGROUND: Until recently, the genetic landscape of small intestinal neuroendocrine tumors (SI-NETs) was limited to recurrent copy number alterations, most commonly a loss on chromosome 18. Intertumor heterogeneity with nonconcordant genotype in paired primary and metastatic lesions also is described, further contributing to the difficulty of unraveling the genetic enigma of SI-NETs. A recent study analyzing 55 SI-NET exomes nominated CDKN1B (p27) as a haploinsufficient tumor suppressor gene. METHODS: This study aimed to determine the frequency of CDKN1B inactivation and to investigate genotype-phenotype correlations. It investigated 362 tumors from 200 patients. All samples were resequenced for mutations in CDKN1B using automated Sanger sequencing. The expression of p27 was investigated in 12 CDKN1B mutant and nine wild type tumors. RESULTS: Some 8.5 % (17/200) of patients had tumors with pathogenic mutations in CDKN1B including 13 insertion deletions, four nonsense variants, and one stop-loss variant. All variants with available nontumoral DNA were classified as somatic. Inter- and intratumor heterogeneity at the CDKN1B locus was detected respectively in six of ten and two of ten patients. Patients with CDKN1B mutated tumors had both heterogeneous disease presentation and diverse prognosis. Expression of the p27 protein did not correlate with CDKN1B mutation status, and no differences in the clinical characteristics between CDKN1B mutated and CDKN1B wild type tumor carriers were found. CONCLUSION: This study corroborates the finding of CDKN1B as a potential haplo-insufficient tumor suppressor gene characterized by inter- and intratumor heterogeneity in SI-NETs.
Assuntos
Biomarcadores Tumorais/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Neoplasias Intestinais/genética , Intestino Delgado/metabolismo , Mutação/genética , Tumores Neuroendócrinos/genética , Adulto , Idoso , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Seguimentos , Heterogeneidade Genética , Humanos , Técnicas Imunoenzimáticas , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Tumores Neuroendócrinos/patologia , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
BACKGROUND: One-fifth of all patients with small-intestinal neuroendocrine tumors (SI-NETs) present with or develop peritoneal carcinomatosis (PC). Our aim was to determine the prognosis and genetic profiles of tumors in patients with PC compared with tumors in patients without PC. METHODS: We included SI-NET patients (cases with PC, n = 73, and controls without PC, n = 468) who underwent operation between 1985 and 2012. The Lyon prognostic index was used to correlate the amount of PC to survival. DNA samples from patients with (n = 8) and without (n = 7) PC were analyzed with a single-nucleotide polymorphism array (HumanOmni2.5 BeadChip, Illumina) to investigate genetic disparities between groups. RESULTS: Patients with PC had poorer survival (median 5.1 years) than controls (11.1 years). An advanced postoperative Lyon prognostic index was a negative prognostic marker for survival by multivariable analysis (P = .042). Patients with and without PC clustered differently based on loss of heterozygosity and copy number variation data from single-nucleotide polymorphism array of the primary tumors (P = .042). CONCLUSION: SI-NET patients with PC have poor survival, which diminishes with increasing PC load after surgery. Clustering based on copy number variation and loss of heterozygosity data suggests different genotypes in primary tumors comparing patients with and without PC.
Assuntos
Carcinoma/secundário , Variação Genética , Neoplasias Intestinais/genética , Tumores Neuroendócrinos/genética , Neoplasias Peritoneais/secundário , Adulto , Idoso , Carcinoma/genética , Carcinoma/mortalidade , Estudos de Coortes , Variações do Número de Cópias de DNA , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Intestinais/mortalidade , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Tumores Neuroendócrinos/mortalidade , Tumores Neuroendócrinos/secundário , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/mortalidade , Prognóstico , Valores de Referência , Estudos Retrospectivos , Medição de Risco , Análise de SobrevidaRESUMO
Adrenal tumors autonomously producing cortisol cause Cushing's syndrome. We performed exome sequencing of 25 tumor-normal pairs and identified 2 subgroups. Eight tumors (including three carcinomas) had many somatic copy number variants (CNVs) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2 and protein-altering mutations in TP53 and RB1. Seventeen tumors (all adenomas) had no somatic CNVs or TP53 or RB1 mutations. Six of these had known gain-of-function mutations in CTNNB1 (ß-catenin) or GNAS (Gαs). Six others had somatic mutations in PRKACA (protein kinase A (PKA) catalytic subunit) resulting in a p.Leu206Arg substitution. Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt Cushing's syndrome). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with the regulatory subunit of PKA, PRKAR1A. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Variações do Número de Cópias de DNA , Hidrocortisona/metabolismo , Mutação , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Exoma , Feminino , Deleção de Genes , Dosagem de Genes , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosforilação , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: About 60% of Pheochromocytoma (PCC) and Paraganglioma (PGL) patients have either germline or somatic mutations in one of the 12 proposed disease causing genes; SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, RET, NF1, TMEM127, MAX and H-RAS. Selective screening for germline mutations is routinely performed in clinical management of these diseases. Testing for somatic alterations is not performed on a regular basis because of limitations in interpreting the results. AIM: The purpose of the study was to investigate genetic events and phenotype correlations in a large cohort of PCC and PGL tumours. METHODS: A total of 101 tumours from 89 patients with PCC and PGL were re-sequenced for a panel of 10 disease causing genes using automated Sanger sequencing. Selected samples were analysed with Multiplex Ligation-dependent Probe Amplification and/or SNParray. RESULTS: Pathogenic genetic variants were found in tumours from 33 individual patients (37%), 14 (16%) were discovered in constitutional DNA and 16 (18%) were confirmed as somatic. Loss of heterozygosity (LOH) was observed in 1/1 SDHB, 11/11 VHL and 3/3 NF1-associated tumours. In patients with somatic mutations there were no recurrences in contrast to carriers of germline mutations (Pâ=â0.022). SDHx/VHL/EPAS1 associated cases had higher norepinephrine output (Pâ=â0.03) and lower epinephrine output (P<0.001) compared to RET/NF1/H-RAS cases. CONCLUSION: Somatic mutations are frequent events in PCC and PGL tumours. Tumour genotype may be further investigated as prognostic factors in these diseases. Growing evidence suggest that analysis of tumour DNA could have an impact on the management of these patients.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Genes Neoplásicos/genética , Paraganglioma/genética , Fenótipo , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/patologia , Sequência de Bases , Estudos de Coortes , Testes Genéticos/métodos , Mutação em Linhagem Germinativa/genética , Humanos , Perda de Heterozigosidade/genética , Dados de Sequência Molecular , Paraganglioma/patologia , Feocromocitoma/patologia , Polimorfismo de Nucleotídeo Único/genética , Estudos Retrospectivos , Análise de Sequência de DNA , SuéciaRESUMO
Pheochromocytoma (PCC) and Paraganglioma are rare tumours originating from neuroendocrine cells. Up to 60% of cases have either germline or somatic mutation in one of eleven described susceptibility loci, SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, RET, NF1, TMEM127 and MYC associated factor-X (MAX). Recently, germline mutations in MAX were found to confer susceptibility to PCC and paraganglioma (PGL). A subsequent multicentre study found about 1% of PCCs and PGLs to have germline or somatic mutations in MAX. However, there has been no study investigating the frequency of MAX mutations in a Scandinavian cohort. We analysed tumour specimens from 63 patients with PCC and PGL treated at Uppsala University hospital, Sweden, for re-sequencing of MAX using automated Sanger sequencing. Our results show that 0% (0/63) of tumours had mutations in MAX. Allele frequencies of known single nucleotide polymorphisms rs4902359, rs45440292, rs1957948 and rs1957949 corresponded to those available in the Single Nucleotide Polymorphism Database. We conclude that MAX mutations remain unusual events and targeted genetic screening should be considered after more common genetic events have been excluded.