Case Report : Micro-RNAs in Plasma From Bilateral Inferior Petrosal Sinus Sampling and Peripheral Blood From Corticotroph Pituitary Neuroendocrine Tumors

Funding Information: The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing infrastructure, biological material and data. Funding Information: This research was funded by the European Regional Development Fund within the project RNA molecular determinants in development of pituitary adenoma” (1.1.1.1/18/A/089). Publisher Copyright: Copyright © 2022 Niedra, Peculis, Konrade, Balcere, Romanovs, Steina, Stukens, Sokolovska, Klovins and Rovite.Objective: Circulating miRNAs are found in bodily fluids including plasma and can serve as biomarkers for diseases. The aim of this study was to provide the first insight into the landscape of circulating miRNAs in close proximity to the adrenocorticotropic hormone (ACTH) secreting PitNET. To achieve this objective next-generation sequencing of miRNAs in plasma from bilateral inferior petrosal sinus sampling (BIPSS) - a gold standard in diagnosing ACTH-secreting PitNETs was carried out and selected miRNA candidates were further tested by RT-qPCR in independent patient cohorts. Methods: Sinistral (left) and dextral (right) BIPSS blood samples of the patient were collected in three time points: before the administration of corticotropin-releasing hormone, 5 and 15 minutes after stimulation. In differential expression analysis, sinistral plasma was compared with dextral. The selected miRNA candidates were tested in plasma by RT-qPCR in two patient groups: 1) in five ACTH secreting PitNET patients with plasma samples taken before and 24 hours after surgery, 2) in 12 ACTH secreting PitNET patients vs. 9 non-functioning PitNET patients. Results: BIPSS concluded that the highest amount of ACTH was released in the sinistral side at the 5th minute mark indicating a presence of a tumor. The highest amount of differentially expressed miRNAs was observed 5 minutes after stimulation (20 upregulated, 14 downregulated). At the 5th minute mark in sinistral plasma, two miRNAs were identified: hsa-miR-7-5p and hsa-miR-375-3p that were highly upregulated compared to other BIPSS samples and peripheral plasma samples. Further testing by qPCR revealed significant reduction of miR-7-5p in plasma 24 hours after surgery and upregulation in plasma of ACTH secreting PitNET patients compared to non-functioning PitNET patients (P =0.0013). Conclusions: By stimulating the ACTH secreting PitNET with CRH a rapid increase of two miRNAs (hsa-mir-7-5p, hsa-mir-375-3p) and ACTH can be observed in sinistral inferior petrosal (tumor side). A decrease of miR-7-5p in plasma after surgery and upregulation in plasma of ACTH secreting PitNET patients was discovered implying that further studies of this miRNA as diagnostic marker is needed.publishersversionPeer reviewe

Whole exome sequencing reveals novel risk genes of pituitary neuroendocrine tumors

Funding Information: This research was supported by the European Regional Development Fund (ERDF), Measure 1.1.1.1 “Industry-Driven Research” project „Molecular markers of pituitary tumour development, progression and therapy response” (Project No. 1.1.1.1/16/A/066, 2017). The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing infrastructure, biological material and data. Publisher Copyright: © 2022 Peculis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Somatic genetic alterations in pituitary neuroendocrine tumors (PitNET) tissues have been identified in several studies, but detection of overlapping somatic PitNET candidate genes is rare. We sequenced and by employing multiple data analysis methods studied the exomes of 15 PitNET patients to improve discovery of novel factors involved in PitNET development. PitNET patients were recruited to the study before PitNET removal surgery. For each patient, two samples for DNA extraction were acquired: venous blood and PitNET tissue. Exome sequencing was performed using Illumina NexSeq 500 sequencer and data analyzed using two separate workflows and variant calling algorithms: GATK and Strelka2. A combination of two data analysis pipelines discovered 144 PitNET specific somatic variants (mean = 9.6, range 0–19 per PitNET) of which all were SNVs. Also, we detected previously known GNAS PitNET mutation and identified somatic variants in 11 genes, which have contained somatic variants in previous WES and WGS studies of PitNETs. Noteworthy, this is the third study detecting somatic variants in gene RYR1 in the exomes of PitNETs. In conclusion, we have identified two novel PitNET candidate genes (AC002519.6 and AHNAK) with recurrent somatic variants in our PitNET cohort and found 13 genes overlapping from previous PitNET studies that contain somatic variants. Our study demonstrated that the use of multiple sequencing data analysis pipelines can provide more accurate identification of somatic variants in PitNETs.publishersversionPeer reviewe

Novel susceptibility loci identified in a genome-wide association study of type 2 diabetes complications in population of Latvia

Funding Information: The study was supported by European Regional Development Fund (ERDF) On Implementation of Activity 1.1.1.2 “Post-doctoral Research Aid” of the Specific Aid Objective 1.1.1 “To increase the research and innovative capacity of scientific institutions of Latvia and the ability to attract external financing, investing in human resources and infrastructure” of the Operational Programme “Growth and Employment”. Project No 1.1.1.2/VIAA/2/18/287 “Identification of clinical subgroups of Type 2 diabetes mellitus and application of pharmacogenetics in the development of personalized antidiabetic therapy”. The funding body was not involved in the design of the study, collection, analysis or interpretation of data, or writing the manuscript. Publisher Copyright: © 2021, The Author(s).Background: Type 2 diabetes complications cause a serious emotional and economical burden to patients and healthcare systems globally. Management of both acute and chronic complications of diabetes, which dramatically impair the quality of patients' life, is still an unsolved issue in diabetes care, suggesting a need for early identification of individuals with high risk for developing diabetes complications. Methods: We performed a genome-wide association study in 601 type 2 diabetes patients after stratifying them according to the presence or absence of four types of diabetes complications: diabetic neuropathy, diabetic nephropathy, macrovascular complications, and ophthalmic complications. Results: The analysis revealed ten novel associations showing genome-wide significance, including rs1132787 (GYPA, OR = 2.71; 95% CI = 2.02–3.64) and diabetic neuropathy, rs2477088 (PDE4DIP, OR = 2.50; 95% CI = 1.87–3.34), rs4852954 (NAT8, OR = 2.27; 95% CI = 2.71–3.01), rs6032 (F5, OR = 2.12; 95% CI = 1.63–2.77), rs6935464 (RPS6KA2, OR = 2.25; 95% CI = 6.69–3.01) and macrovascular complications, rs3095447 (CCDC146, OR = 2.18; 95% CI = 1.66–2.87) and ophthalmic complications. By applying the targeted approach of previously reported susceptibility loci we managed to replicate three associations: MAPK14 (rs3761980, rs80028505) and diabetic neuropathy, APOL1 (rs136161) and diabetic nephropathy. Conclusions: Together these results provide further evidence for the implication of genetic factors in the development of type 2 diabetes complications and highlight several potential key loci, able to modify the risk of developing these conditions. Moreover, the candidate variant approach proves a strong and consistent effect for multiple variants across different populations.publishersversionPeer reviewe

Polymorphisms in MEN1 and DRD2 genes are associated with the occurrence and characteristics of pituitary adenomas

Publisher Copyright: © 2016 European Society of Endocrinology Published by Bioscientifica Ltd. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.Objective: Although pituitary adenomas (PAs) affect a significant proportion of the population, only a fraction have the potential to become clinically relevant during an individual's lifetime, causing hormonal imbalance or complications due to mass effect. The overwhelming majority of cases are sporadic and without a clear familial history, and the genotype-phenotype correlation in PA patients is poorly understood. Our aim was to investigate the involvement of genes known for their role in familial cases on drug response and tumor suppression in the development and pathology of PAs in a patient group from Latvia. Design: The study included 143 cases and 354 controls, we investigated the role of single-nucleotide polymorphisms (SNPs) in seven genes (SSTR2, SSTR5, DRD2, MEN1, AIP, GNAS, and PRKAR1A) associated with pituitary tumor occurrence, phenotype, and clinical symptoms. Methods: Genotyping of 96 tag and nonsynonymous SNPs was performed in the genomic regions of interest. Results: We discovered a significant association (OR = 17.8, CI 0.95 = 2.18-145.5, P = 0.0002) between a rare MEN1 mutation (rs2959656) and clinically active adenoma in our patients. Additionally, rs7131056 at DRD2 was associated with a higher occurrence of extrasellar growth in patients with prolactinoma and somatotropinoma (OR = 2.79, CI 0.95 = 1.58-4.95, P = 0.0004). Conclusions: rs2959656, a nonsynonymous variant in MEN1, is associated with the development of clinically active PA. Furthermore, rs7131056 in DRD2 contributes to either faster growth of the adenoma or reduced symptomatic presentation, allowing PAs to become larger before detection.publishersversionPeer reviewe

Evaluation of the Possibility to Detect Circulating Tumor DNA From Pituitary Adenoma

Funding Information: The authors acknowledge the Latvian Biomedical Research and Study Center and the Genome Database of the Latvian Population for providing infrastructure, biological material, and data. Funding. This research was supported by the European Regional Development Fund (ERDF), Measure 1.1.1.1 Industry-Driven Research project Molecular markers of pituitary tumor development, progression and therapy response (Project No. 1.1.1.1/16/A/066, 2017), and Roche Academy project Immunohistochemical analysis of pituitary adenoma tissue. Publisher Copyright: © Copyright © 2019 Megnis, Peculis, Rovite, Laksa, Niedra, Balcere, Caune, Breiksa, Nazarovs, Stukens, Konrade, Pirags and Klovins.Objective: Circulating free DNA (cfDNA) in general and circulating tumor DNA (ctDNA) in particular is becoming an increasingly used form of liquid biopsy biomarkers. In this study, we are investigating the ability to detect ctDNA from the plasma of pituitary adenoma (PA) patients. Design: Tumor tissue samples were obtained from planed PA resections, before which blood plasma samples were taken. Somatic variants found in PA tissue samples were evaluated in related cfDNA, isolated from plasma samples. Methods: Sanger sequencing, as well as previously obtained whole-exome sequencing data, were used to evaluate somatic variants composition in tumor tissue samples. cfDNA was isolated from the same PA patients and competitive allele-specific TaqMan PCR and amplicon-based next-generation sequencing (NGS) approach were used for targeted detection of variants found in corresponding tumor tissue samples. Results: Using NGS-based analysis, we detected five out of 17 somatic variants in 40 to 60% of total reads, three variants in 0.50–5.00% of total read count, including GNAS c.601C>T, which was detected using ultra-deep NGS (1.78 million X) in 0.77% of amplicons reads. Nine variants were not detected. We also detected We were not able to detect variant found in PA tissue in cfDNA using cast-PCR, indicating that the portion of variant-containing ctDNA in total isolated cfDNA is too small to be detected with this method. Conclusions: For the first time, we demonstrate the possibility to detect somatic variants of PA in cfDNA isolated from patients' blood plasma. Whether the source of variant detected in cfDNA is PA should be further tested.publishersversionPeer reviewe

Genome wide analysis of circulating miRNAs in growth hormone secreting pituitary neuroendocrine tumor patients’ plasma

Funding Information: The financial support given by NSFC with Grant No. 11874034, Taishan Scholar Program of Shandong No. ts201712054, and Shandong Science and Technology Enterprise Innovation Capacity Enhancement Project (2021TSGC1036) are highly appreciated for this research. Publisher Copyright: Copyright © 2022 Niedra, Peculis, Litvina, Megnis, Madrika, Balcere, Romanovs, Steina, Stukens, Breiksa, Nazarovs, Sokolovska, Liutkeviciene, Vilkevicute, Konrade and Rovite.Background: Circulating plasma miRNAs have been increasingly studied in the field of pituitary neuroendocrine tumor (PitNET) research. Our aim was to discover circulating plasma miRNAs species associated with growth hormone (GH) secreting PitNETs versus assess how the plasma levels of discovered miRNA candidates are impacted by SSA therapy and whether there is a difference in their levels between GH secreting PitNETs versus other PitNET types and healthy individuals. Design: We compared plasma miRNA content and levels before and after surgery focusing on GH secreting PitNET patients. Selected miRNA candidates from our data and literature were then tested in a longitudinal manner in somatostatin analogues (SSA) treatment group. Additionally, we validated selected targets in an independent GH secreting PitNET group. Methods: miRNA candidates were discovered using the whole miRNA sequencing approach and differential expression analysis. Selected miRNAs were then analyzed using real-time polymerase chain reaction (qPCR). Results: Whole miRNA sequencing discovered a total of 16 differentially expressed miRNAs (DEMs) in GH secreting PitNET patients’ plasma 24 hours after surgery and 19 DEMs between GH secreting PitNET patients’ plasma and non-functioning (NF) PitNET patients’ plasma. Seven miRNAs were selected for further testing of which miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p showed a significant downregulation in plasma after 1 month of SSA treatment. mir-625-5p was found to be significantly downregulated in plasma of GH secreting PitNET patients vs. NF PitNET patients. miR-625-5p alongside miR-130b-3p were also found to be downregulated in GH PitNETs compared to healthy individuals. Conclusions: Our study suggests that expression of plasma miRNAs miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p in GH secreting PitNETs is affected by SSA treatment. Additionally, miR-625-5p can distinguish GH secreting PitNETs from other PitNET types and healthy controls warranting further research on these miRNAs for treatment efficacy.publishersversionPeer reviewe

Significantly altered peripheral blood cell DNA methylation profile as a result of immediate effect of metformin use in healthy individuals

Funding Information: The work was supported by the European Regional Development Fund under the project “Investigation of interplay between multiple determinants influencing response to metformin: search for reliable predictors for efficacy of type 2 diabetes therapy” (Project Nr.: 1.1.1.1/16/A/091). Publisher Copyright: © 2018 The Author(s).Background: Metformin is a widely prescribed antihyperglycemic agent that has been also associated with multiple therapeutic effects in various diseases, including several types of malignancies. There is growing evidence regarding the contribution of the epigenetic mechanisms in reaching metformin's therapeutic goals; however, the effect of metformin on human cells in vivo is not comprehensively studied. The aim of our study was to examine metformin-induced alterations of DNA methylation profiles in white blood cells of healthy volunteers, employing a longitudinal study design. Results: Twelve healthy metformin-naïve individuals where enrolled in the study. Genome-wide DNA methylation pattern was estimated at baseline, 10 h and 7 days after the start of metformin administration. The whole-genome DNA methylation analysis in total revealed 125 differentially methylated CpGs, of which 11 CpGs and their associated genes with the most consistent changes in the DNA methylation profile were selected: POFUT2, CAMKK1, EML3, KIAA1614, UPF1, MUC4, LOC727982, SIX3, ADAM8, SNORD12B, VPS8, and several differentially methylated regions as novel potential epigenetic targets of metformin. The main functions of the majority of top-ranked differentially methylated loci and their representative cell signaling pathways were linked to the well-known metformin therapy targets: regulatory processes of energy homeostasis, inflammatory responses, tumorigenesis, and neurodegenerative diseases. Conclusions: Here we demonstrate for the first time the immediate effect of short-term metformin administration at therapeutic doses on epigenetic regulation in human white blood cells. These findings suggest the DNA methylation process as one of the mechanisms involved in the action of metformin, thereby revealing novel targets and directions of the molecular mechanisms underlying the various beneficial effects of metformin. Trial registration: EU Clinical Trials Register, 2016-001092-74. Registered 23 March 2017, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001092-74/LV.Peer reviewe

Medication for Acromegaly Reduces Expression of MUC16, MACC1 and GRHL2 in Pituitary Neuroendocrine Tumour Tissue

Funding Information: The authors acknowledge the Latvian Biomedical Research and Study Centre and the Genome Database of the Latvian Population for providing the infrastructure, biological material, and data. Publisher Copyright: © Copyright © 2021 Saksis, Silamikelis, Laksa, Megnis, Peculis, Mandrika, Rogoza, Petrovska, Balcere, Konrade, Steina, Stukens, Breiksa, Nazarovs, Sokolovska, Pirags, Klovins and Rovite.Acromegaly is a disease mainly caused by pituitary neuroendocrine tumor (PitNET) overproducing growth hormone. First-line medication for this condition is the use of somatostatin analogs (SSAs), that decrease tumor mass and induce antiproliferative effects on PitNET cells. Dopamine agonists (DAs) can also be used if SSA treatment is not effective. This study aimed to determine differences in transcriptome signatures induced by SSA/DA therapy in PitNET tissue. We selected tumor tissue from twelve patients with somatotropinomas, with half of the patients receiving SSA/DA treatment before surgery and the other half treatment naive. Transcriptome sequencing was then carried out to identify differentially expressed genes (DEGs) and their protein–protein interactions, using pathway analyses. We found 34 upregulated and six downregulated DEGs in patients with SSA/DA treatment. Three tumor development promoting factors MUC16, MACC1, and GRHL2, were significantly downregulated in therapy administered PitNET tissue; this finding was supported by functional studies in GH3 cells. Protein–protein interactions and pathway analyses revealed extracellular matrix involvement in the antiproliferative effects of this type of the drug treatment, with pronounced alterations in collagen regulation. Here, we have demonstrated that somatotropinomas can be distinguished based on their transcriptional profiles following SSA/DA therapy, and SSA/DA treatment does indeed cause changes in gene expression. Treatment with SSA/DA significantly downregulated several factors involved in tumorigenesis, including MUC16, MACC1, and GRHL2. Genes that were upregulated, however, did not have a direct influence on antiproliferative function in the PitNET cells. These findings suggested that SSA/DA treatment acted in a tumor suppressive manner and furthermore, collagen related interactions and pathways were enriched, implicating extracellular matrix involvement in this anti-tumor effect of drug treatment.publishersversionPeer reviewe

Single nucleotide polymorphisms in the intergenic region between metformin transporter OCT2 and OCT3 coding genes are associated with short-Term response to metformin monotherapy in type 2 diabetes mellitus patients

Publisher Copyright: © 2016 The authors Published by Bioscientifica Ltd. Copyright: Copyright 2017 Elsevier B.V., All rights reserved.Objective(s): High variability in clinical response to metformin is often observed in type 2 diabetes (T2D) patients, and it highlights the need for identification of genetic components affecting the efficiency of metformin therapy. Aim of this observational study is to evaluate the role of tagSNPs (tagging single nucleotide polymorphisms) from genomic regions coding for six metformin transporter genes with respect to the short-Term efficiency. Design: 102 tagSNPs in 6 genes coding for metformin transporters were genotyped in the group of 102 T2D patients treated with metformin for 3 months. Methods: Most significant hits were analyzed in the group of 131 T2D patients from Slovakia. Pharmacokinetic study in 25 healthy nondiabetic volunteers was conducted to investigate the effects of identified polymorphisms. Results: In the discovery group of 102 patients, minor alleles of rs3119309, rs7757336 and rs2481030 were significantly nominally associated with metformin inefficiency (P = 1.9 × 106 to 8.1 × 106). Effects of rs2481030 and rs7757336 did not replicate in the group of 131 T2DM patients from Slovakia alone, whereas rs7757336 was significantly associated with a reduced metformin response in combined group. In pharmacokinetic study, group of individuals harboring risk alleles of rs7757336 and rs2481030 displayed significantly reduced AUC∞ of metformin in plasma. Conclusions: For the first time, we have identified an association between the lack of metformin response and SNPs rs3119309 and rs7757336 located in the 5 flanking region of the genes coding for Organic cation transporter 2 and rs2481030 located in the 5 flanking region of Organic cation transporter 3 that was supported by the results of a pharmacokinetic study on 25 healthy volunteers.publishersversionPeer reviewe