Next-generation sequencing identifies novel mitochondrial variants in pituitary adenomas.

PURPOSE: Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants. We aimed to investigate the mitochondrial genome in pituitary adenomas by NGS. METHODS: We analysed 11 growth hormone producing and 33 non-functioning [22 gonadotroph and 11 hormone immunonegative] pituitary adenomas using VariantPro™ Mitochondrion Panel on Illumina MiSeq instrument. Revised Cambridge Reference Sequence (rCRS) of the mtDNA was used as reference. Heteroplasmy was determined using a 3% cutoff. RESULTS: 496 variants were identified in pituitary adenomas with overall low level of heteroplasmy (7.22%). On average, 35 variants were detected per sample. Samples harbouring the highest number of variants had the highest Ki-67 indices independently of histological subtypes. We identified eight variants (A11251G, T4216C, T16126C, C15452A, T14798C, A188G, G185A, and T16093C) with different prevalences among different histological groups. T16189C was found in 40% of non-recurrent adenomas, while it was not present in the recurrent ones. T14798C and T4216C were confirmed by Sanger sequencing in all 44 samples. 100% concordance was found between NGS and Sanger method. CONCLUSIONS: NGS is a reliable method for investigating mitochondrial genome and heteroplasmy in pituitary adenomas. Out of the 496 detected variants, 414 have not been previously reported in pituitary adenoma. The high number of mtDNA variants may contribute to adenoma genesis, and some variants (i.e., T16189C) might associate with benign behaviour.

Limitations of high throughput methods for miRNA expression profiles in non-functioning pituitary adenomas.

Microarray, RT-qPCR based arrays and next-generation-sequencing (NGS) are available high-throughput methods for miRNA profiling (miRNome). Analytical and biological performance of these methods were tested in identification of biologically relevant miRNAs in non-functioning pituitary adenomas (NFPA). miRNome of 4 normal pituitary (NP) and 8 NFPA samples was determined by these platforms and expression of 21 individual miRNAs was measured on 30 (20 NFPA and 10 NP) independent samples. Complex bioinformatics was used. 132 and 137 miRNAs were detected by all three platforms in NP and NFPA, respectively, of which 25 were differentially expressed (fold change > 2). The strongest correlation was observed between microarray and TaqMan-array, while the data obtained by NGS were the most discordant despite of various bioinformatics settings. As a technical validation we measured the expression of 21 selected miRNAs by individual RT-qPCR and we were able to validate 35.1%, 76.2% and 71.4% of the miRNAs revealed by SOLiD, TLDA and microarray result, respectively. We performed biological validation using an extended number of samples (20 NFPAs and 8 NPs). Technical and biological validation showed high correlation (p < 0.001; R = 0.96). Pathway and network analysis revealed several common pathways but no pathway showed the same activation score. Using the 25 platform-independent miRNAs developmental pathways were the top functional categories relevant for NFPA genesis. The difference among high-throughput platforms is of great importance and selection of screening method can influence experimental results. Validation by another platform is essential in order to avoid or to minimalize the platform specific errors.