Comprehensive genomic analysis of adrenocortical carcinoma reveals genetic profiles associated with patient survival.

BACKGROUND: Adrenocortical carcinoma (ACC) is one of the most lethal endocrine malignancies and there is a lack of clinically useful markers for prognosis and patient stratification. Therefore our aim was to identify clinical and genetic markers that predict outcome in patients with ACC. METHODS: Clinical and genetic data from a total of 162 patients with ACC were analyzed by combining an independent cohort consisting of tumors from Yale School of Medicine, Karolinska Institutet, and Düsseldorf University (YKD) with two public databases [The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO)]. We used a novel bioinformatical pipeline combining differential expression and messenger RNA (mRNA)- and DNA-dependent survival. Data included reanalysis of previously conducted whole-exome sequencing (WES) for the YKD cohort, WES and RNA data for the TCGA cohort, and RNA data for the GEO cohort. RESULTS: We identified 3903 significant differentially expressed genes when comparing ACC and adrenocortical adenoma, and the mRNA expression levels of 461/3903 genes significantly impacted survival. Subsequent analysis revealed 45 of these genes to be mutated in patients with significantly worse survival. The relationship was significant even after adjusting for stage and age. Protein-protein interaction showed previously unexplored interactions among many of the 45 proteins, including the cancer-related proteins DNA polymerase delta 1 (POLD1), aurora kinase A (AURKA), and kinesin family member 23 (KIF23). Furthermore 14 of the proteins had significant interactions with TP53 which is the most frequently mutated gene in the germline of patients with ACC. CONCLUSIONS: Using a multiparameter approach, we identified 45 genes that significantly influenced survival. Notably, many of these genes have protein interactions not previously implicated in ACC. These findings may lay the foundation for improved prognostication and future targeted therapies.

[Hyperaldosteronism]. / Hyperaldosteronismus.

Aldosterone is produced in the adrenal cortex and governs volume and electrolyte homeostasis. Hyperaldosteronism can occur either as primary aldosteronism (renin-independent) or secondary aldosteronism (renin-dependent). As the commonest cause of secondary hypertension, primary aldosteronism is associated with increased cardiovascular risk. Its most prevalent subtypes are aldosterone-producing adenomas as the most frequent unilateral form and bilateral hyperaldosteronism. Unilateral hyperplasia, familial hyperaldosteronism and aldosterone-producing carcinoma are rare. The aldosterone/renin ratio serves as a screening parameter for primary aldosteronism. If this ratio is elevated, confirmatory testing and adrenal imaging are performed. Adrenal venous sampling is considered the gold standard for the distinction of unilateral from bilateral disease. Unilateral disease can potentially be cured by adrenalectomy, whereas patients that are not candidates for surgery or have bilateral disease are treated with mineralocorticoid receptor antagonists. Over the past 10 years, somatic mutations in ion channels or transporters have been identified as causes of aldosterone-producing adenomas and so-called aldosterone-producing cell clusters (potential precursors of adenomas and correlates of bilateral hyperplasia, but also of subclinical hyperaldosteronism). In addition, germline mutations in overlapping genes cause familial hyperaldosteronism. Secondary hyperaldosteronism can occur in patients with hypertension treated with diuretics or in renal artery stenosis.

[Primary aldosteronism : Genetics and pathology]. / Primärer Hyperaldosteronismus : Genetik und Pathologie.

BACKGROUND: Primary aldosteronism, the excessive production of the steroid hormone aldosterone, is the most common cause of secondary hypertension. Common subforms include bilateral adrenal hyperplasia and aldosterone-producing adenoma. OBJECTIVES: The goal of this review is to summarize important publications on the genetic basis of primary aldosteronism. RESULTS: Somatic mutations in the KCNJ5, CACNA1D, ATP1A1, and ATP2B3 genes have been described as causes of aldosterone-producing adenomas. They eventually all lead to increased cellular calcium influx and aldosterone production. The mechanisms of rare CTNNB1 mutations are less defined. Correlations between mutations and different histologic characteristics as well as gender and ethnicity remain unexplained. Recent publications suggest that bilateral hyperplasia is at least partially due to so-called aldosterone-producing cell clusters, often with mutations in CACNA1D. Rare familial forms show mutations in the CYP11B2, CLCN2, KCNJ5, CACNA1H, or CACNA1D genes. CONCLUSIONS: These results suggest that a significant fraction of primary aldosteronism is due to somatic mutations in single genes.

An Update on Familial Hyperaldosteronism.

Familial forms of primary aldosteronism have been suggested to account for up to 6% of cases in referral centers. For many years, the genetics of familial hyperaldosteronism remained unknown, with the notable exception of glucocorticoid-remediable aldosteronism, due to unequal crossing over and formation of a chimeric 11ß-hydroxylase/aldosterone synthase gene. Over the past 5 years, mutations in 3 additional genes have been shown to cause familial forms of primary aldosteronism. Gain-of-function heterozygous germline mutations in KCNJ5, which encodes an inward rectifier potassium channel, cause autosomal dominant syndromes of PA and hypertension with or without adrenal hyperplasia. Germline mutations in CACNA1D, which codes for an L-type calcium channel, have so far only been found in 2 cases with a syndrome of primary aldosteronism, seizures, and neurologic abnormalities. Both KCNJ5 and CACNA1D mutations in familial hyperaldosteronism were only discovered following identification of similar or identical somatic mutations in aldosterone-producing adenomas. In contrast, a recent exome sequencing study identified germline mutations in CACNA1H (a T-type calcium channel), previously undescribed in adenomas, in 5 unrelated families with early-onset primary aldosteronism and hypertension, without any additional shared symptoms. Future exome or genome sequencing studies are expected to shed light on the genetic basis of many cases of familial hyperaldosteronism that remain unexplained.

Potentiation of in vivo neuroprotection by BclX(L) and GDNF co-expression depends on post-lesion time in deafferentiated CNS neurons.

To elucidate effective and long-lasting neuroprotective strategies, we analysed a combination of mitochondrial protection and neurotrophic support in two well-defined animal models of neurodegeneration, traumatic lesion of optic nerve and complete 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal pathway. Neuroprotection by BclX(L), Glial cell line-derived neurotrophic factor (GDNF) or BclX(L) plus GDNF co-expression were studied at 2 weeks and at 6-8 weeks after lesions. In both lesion paradigms, the efficacy of this combination approach significantly differed depending on post-lesion time. We show that BclX(L) expression is more important for neuronal survival in the early phase after lesions, whereas GDNF-mediated neuroprotection becomes more prominent in the advanced state of neurodegeneration. BclX(L) expression was not sufficient to finally inhibit degeneration of deafferentiated central nervous system neurons. Long-lasting GDNF-mediated neuroprotection depended on BclX(L) co-expression in the traumatic lesion paradigm, but was independent of BclX(L) in the 6-OHDA lesion model. The results demonstrate that neuroprotection studies in animal models of neurodegenerative diseases should generally be performed over extended periods of time in order to reveal the actual potency of a therapeutic approach.

Evaluation of epitope tags for protein detection after in vivo CNS gene transfer.

Functional characterization of disease-related proteins, their splice variants and dominant negative mutants in the context of complex CNS tissues such as brain and retina is frequently assessed by in vivo gene transfer. For correct interpretation of results it is imperative that the protein under investigation is unambiguously detected in the transduced cell types and can be distinguished from any endogenously expressed physiological variants. Therefore the first systematic evaluation of epitope tags used to trace ectopically expressed proteins in the central nervous system is presented here. Substantial differences in the performances of various epitope tag-antibody combinations with respect to sensitivity, specificity and influence of the epitope tag on the fusion protein are elucidated. Epitope tags already established for protein detection in vitro and to some extent in vivo (c-Myc, HA and FLAG tags) were immunohistochemically detected with high sensitivity. However, detection of these tags revealed problems with background staining and we also document structural and functional influence of the tags on the fusion protein. In order to prevent such unwanted side-effects, epitope tags which have not yet been used for in vivo applications (IRS, EE and AU1 tags) were characterized in brain, retina and cultured neurons. While use of the IRS and EE tags was hindered by low sensitivity or specificity, optimal results were obtained with the AU1 epitope, which may develop into a standard tool for detection of ectopic protein expression in the central nervous system.

Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units.

Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology.

Characterization of the human T cell response against the neuronal protein synapsin in patients with multiple sclerosis.

Although multiple sclerosis (MS) is considered primarily as a demyelinating disease, neuronal damage is abundant and correlates with the neurological deficit. Therefore, we investigated the frequency and characteristics of human T cells specific for synapsin-a neuronal protein highly conserved among species. Synapsin specific T cell responses were detected at a frequency similar to that of MBP specific T cells in MS patients, one patient with acute demyelinating encephalomyelitis (ADEM) and controls. Long-term T cell lines specific for synapsin exhibited a CD3(+), CD4(+), CD8(-) phenotype and produced high amounts of tumor-necrosis-factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) after antigen specific stimulation, whereas lymphotoxin (LT), interleukin-4 (IL-4) and interleukin-10 (IL-10) were detectable in smaller quantities.

Glatiramer acetate (copolymer-1)-specific, human T cell lines: cytokine profile and suppression of T cell lines reactive against myelin basic protein.

Glatiramer acetate (GA), represents an established treatment of relapsing/remitting multiple sclerosis (MS). The mechanisms responsible for the effect of GA are not fully understood. We generated GA-, myelin basic protein (MBP)- and purified protein derivative (PPD)-specific T cell lines from three MS patients and one healthy donor. The GA-specific lines were CD3+, CD4+, CD8- and produced tumor-necrosis-factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), interleukin-6 (IL-6) and interleukin-10 (IL-10) after stimulation with GA in the presence of irradiated peripheral blood mononuclear cells. MBP-specific T cell lines showed an identical phenotype and secreted TNF-alpha, IFN-gamma, IL-4, IL-10, but not IL-6. Co-culture experiments demonstrated, that GA-specific T cell lines have the capability to suppress the proliferation of MBP-specific T cell lines.