Comparison of adipose tissue derived genes in endogenous Cushing’s syndrome versus diet-induced obesity

Introduction: Dysregulation of adipokine secretion and action is a characteristic feature of obesity and a key clinical feature of Cushing’s syndrome (CS). We have investigated whether endogenous glucocorticoid excess influences adipose tissue-derived gene expression. Material and methods: mRNA expression of adipokines; adiponectin, resistin, tumour necrosis factor-a, interleukin-6 (IL-6), angiotensinogen (AGT), plasminogen activator inhibitor type 1, retinol binding protein 4, visfatin, and cystatin C was assessed by quantitative real-time RT-PCR in visceral adipose tissue removed during abdominal surgery of eight patients with CS, and six control patients. Results: We did not find any significant difference in the investigated genes; however, the almost significant overexpression of AGT and underexpression of IL-6 might be noteworthy (p = 0.06 in both cases). Conclusions: No significant differences were found in the expression of the investigated genes known as cardiometabolic risk factors. This indicates that there are no major differences between endogenous hypercortisolism or diet-induced obesity regarding the expression of adipokines involved in cardiometabolic disorders. However, the difference in AGT and IL-6 expression might be included in pathways affecting fat distribution in C

Circulating miRNA Expression Profiling in Primary Aldosteronism

Objective: Primary aldosteronism is a major cause of secondary hypertension. Its two principal forms are bilateral adrenal hyperplasia (BAH) and aldosterone-producing adenoma (APA) whose differentiation is clinically pivotal. There is a major clinical need for a reliable and easily accessible diagnostic biomarker for case identification and subtyping. Circulating microRNAs were shown to be useful as minimally invasive diagnostic markers. Our aim was to determine and compare the circulating microRNA expression profiles of adenoma and hyperplasia plasma samples, and to evaluate their applicability as minimally invasive markers. Methods: One hundred and twenty-three samples from primary aldosteronism patients were included. Next-generation sequencing was performed on 30 EDTA-anticoagulated plasma samples (discovery cohort). Significantly differently expressed miRNAs were validated by real-time reverse transcription-qPCR in an independent validation cohort (93 samples). Results: We have found relative overexpression of miR-30e-5p, miR-30d-5p, miR-223-3p, and miR-7-5p in hyperplasia compared to adenoma by next-generation sequencing. Validation by qRT-PCR confirmed significant overexpression of hsa-miR-30e-5p, hsa-miR-30d-5p, and hsa-miR-7-5p in hyperplasia samples. Regarding the microRNA expressional variations, adenoma is more heterogeneous at the miRNA level compared to hyperplasia. Conclusion: Three microRNAs were significantly overexpressed in hyperplasia samples compared to adenoma samples, but their sensitivity and specificity values are not good enough for introduction to clinical practice

Heterogeneous genetic background of Hungarian patients with pheochromocytoma/paraganglioma requires gene panel testing

Introduction Pheochromocytomas and paragangliomas (Pheo/PGL) are rare neuroendocrine tumours arising from the adrenal medulla or the symphathetic paraganglia, respectively. Germline mutations are present in w40% of the patients. To date, at least 16 genes have been demonstrated to be involved in the genetic background of Pheo/PGL. Prioritization in order of genes tested can be applied, but if the probability of a disease-associated germline mutation exceeds 10% the testing of all susceptibility genes is recommended. Using next generation sequencing (NGS) based methods for genetic testing of Pheo/PGL associated genes progressively becomes part of the routine diagnostics. Objective To assess the genetic background of Hungarian patients with Pheo/PGL and to develop a NGS based gene panel assay for analysis of Pheo/PGL susceptibility genes. Methods We examined 131 patients with the diagnosis of Pheo/PGL diagnosed and nursed at the 2nd Department of Medicine, Semmelweis University. The prevalence of the germline mutations of Pheo/PGL genes was determined using conventional methods. Genotype-phenotype correlations were evaluated. A gene panel covering 15 genes (RET, VHL, NF1, EPAS, EGLN1, KIF1B, SDHA, SDHB, SDHAF2, SDHC, SDHD, FH, MAX, TMEM127, MEN1) was developed and analytical sensitivity was evaluated on 36 patients with known genetic background. Library preparation was performed using SeqCapEZ capture platform with our probe design. Illumina MiSeq instrument was used for sequencing. Sequencing data were analysed with GATK workflow. Variant annotation was performed with SNPeffect. Results Germline mutations of Pheo/PGL genes were present in at 34% of the patients: 10 (7.6%) SDHB, 9 (6.9%) RET, 5 (3.8%) VHL, TMEM127, MDH2, 4 (3%) NF1, 3 (2.3%) SDHD, 2 (1.5%) SDHC and KIF1B. 5 of 10 SDHB mutation carriers developed malignant disease. Homozygous form of a MDH2 variant was associated with malignancy. Among the 10 patients with bilateral adrenal Pheo 4 RET, 2 TMEM127 and 1 VHL mutations were identified. The coverage of genes in our panel was higher than 150 reads in all regions and all known mutations were correctly identified. Discussion Our findings regarding the prevalence of germline mutations in the development of Pheo/PGL are in accordance with the literature. No founder mutation occurred in our population as we could detect mutations in 9 genes, underlining the need of novel methods for mutation analysis in everyday clinical practice. Our NGSbased gene panel performed accurately, however two recently identified genes (MDH2, GOT2) were not covered

Next generation sequencing for characterization of mitochondrial genome in pituitary adenomas

Introduction Disrupted mitochondrial functions and genetic variations of mitochondrial DNA (mtDNA) have been observed in different tumors. Regarding pituitary adenomas mtDNA was evaluated only in oncocytic type using PCR based methods and it showed high prevalence of Complex I variants. Next generation sequencing (NGS) allows high throughput sequencing and it is useful for accurate identification of heteroplasmy of mitochondrial genome as well. Aim We aimed to investigate the entire mitochondrial genome in different adenoma types. Material and methods We collected 22 gonadotroph (GO), 11 growth hormone producing (GH) and 11 null-cell (NC) adenoma specimens from samples removed by transsphenoidal surgery. From fresh frozen tissues DNA extraction was performed using QIAamp Fast DNA Tissue Kit. For library preparation VariantPro™ Amplicon Mitochondrion Panel kit was used. The total mtDNA (16569 bp) was sequenced on Illumina MiSeq Instrument. Following complex bioinformatic analysis Revised Cambridge Reference Sequence (rCRS) of the human mitochondrial DNA was used as reference. Heteroplasmy was determined using 3% cutoff. Results. The whole mitochondrial genome were covered by 630±370 (avg±SE) reads per base. 496 variants were identified in adenomas compared to reference sequence. Overall a low (7.22%) heteroplasmy prevalence was found. Based on mitochondrial sequence variants by hierarchical cluster analysis we could not discriminate different adenoma types. No association between Ki-67 index or recurrent-nonrecurrent status of adenomas and mitochondrial variants were detected. Four variants appeared more often in null-cell adenomas compared to gonadotroph adenomas (chrM_188: 18% vs. 0%, chrM_16093: 18% vs. 0%, chrM_185: 27% vs. 0% and chrM_14798: 36% vs. 5%; Padj=0.0246, 0.0246, 0.01542 and 0.01829, respectively). Of these variants chrM_14798, chrM_4216 and chrM_15452 are non-synonymous polymorphisms leading to amino acid change in MT-CYB (mitochondrially encoded cytochrome b) and in MT-ND1 (mitochondrially encoded NADH dehydrogenase 1) genes. We identified chrM_16189 variant (non-protein coding variant) in 40% (6/15) of nonrecurrent adenomas compared to recurrent ones where this variant was not present (0/11) (p=0.0209). Conclusions Next-generation sequencing is a reliable method for investigating mitochondrial genome and heteroplasmy in pituitary adenomas. In pituitary adenomas the prevalence of heteroplasmy of mitochondrial genome is low suggesting that these alterations may not influence mitochondrial function considerably. Of pituitary tumours only null cell adenomas possess alterations of mitochondrial genome with potential functional consequences suggesting that during the development of this subtype of pituitary tumours mitochondrial function-associated mechanisms may have role