Promocijas darbs

Elektroniskā versija nesatur pielikumusDarbā analizētas hipofīzes šūnu kultūru biomolekulārās īpašības un no hipofīzes izcelsmes šūnām brīvās DNS molekulu klātbūtne pacientu asins plazmā. Tika konstatēts, ka viena no šūnu kultūrām ir multipotentas mezinhimāli stromālās šūnas, kas visticamāk nav galvenā PitNET šūnu populācija, bet, iespējams, tai ir audzēja darbību atbalstoša loma. Otra šūnu kultūra ir hipofīzes sfēru veidojošas šūnas, kuras visticamāk, veido audzēja galveno audu masu. Tām bija kopīgas ģenētiskās iezīmes ar galveno audzēja masu. Analizējot pacientu šķidrās biopsijas, tika atklāts, ka PitNET audu paraugos atrastās somatiskās mutācijas ar dažādu jutību ir konstatējamas arī šūnu brīvajā DNS. Atslēgvārdi: hipofīzes neiroendokrīnie audzēji, neparenhimālās šūnas, multipotentas mezinhimāli stromālās šūnas, hipofīzes sfēras, šūnu brīvā DNS, cirkulējošā audzēja DNSThe thesis analyses biomolecular signatures of pituitary cell cultures and the representation of pituitary-borne cell-free DNA molecules in the patients’ blood plasma. One of the cell cultures was detected to be multipotent mesenchymal stromal cells, which are not the main PitNET cell population but likely have a tumour supportive role. The other cell culture was pituitary sphere-forming cells, which likely constitute the main tissue mass of the tumour. They shared genetic traits with the main tumour mass. Analysing patients’ liquid biopsies revealed that the somatic mutations found in PitNET tissue samples are also detectable in cell-free DNA with varying sensitivity. Keywords: pituitary neuroendocrine tumours, non-parenchymal cells, multipotent mesenchymal stromal cells, pituispheres, cell-free DNA, circulating tumour DN

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

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

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

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

First Report on the Latvian SARS-CoV-2 Isolate Genetic Diversity

Copyright © 2021 Zrelovs, Ustinova, Silamikelis, Birzniece, Megnis, Rovite, Freimane, Silamikele, Ansone, Pjalkovskis, Fridmanis, Vilne, Priedite, Caica, Gavars, Perminov, Storozenko, Savicka, Dimina, Dumpis and Klovins.Remaining a major healthcare concern with nearly 29 million confirmed cases worldwide at the time of writing, novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 920 thousand deaths since its outbreak in China, December 2019. First case of a person testing positive for SARS-CoV-2 infection within the territory of the Republic of Latvia was registered on 2nd of March 2020, 9 days prior to the pandemic declaration by WHO. Since then, more than 277,000 tests were carried out confirming a total of 1,464 cases of coronavirus disease 2019 (COVID-19) in the country as of 12th of September 2020. Rapidly reacting to the spread of the infection, an ongoing sequencing campaign was started mid-March in collaboration with the local testing laboratories, with an ultimate goal in sequencing as much local viral isolates as possible, resulting in first full-length SARS-CoV-2 isolate genome sequences from the Baltics region being made publicly available in early April. With 133 viral isolates representing ~9.1% of the total COVID-19 cases during the "first coronavirus wave" in the country (early March, 2020-mid-September, 2020) being completely sequenced as of today, here, we provide a first report on the genetic diversity of Latvian SARS-CoV-2 isolates.publishersversionPeer reviewe

Novel Insights into Pituitary Tumorigenesis: Genetic and Epigenetic Mechanisms.

Substantial advances have been made recently in the pathobiology of pituitary tumors. Similar to many other endocrine tumors, over the last few years we have recognized the role of germline and somatic mutations in a number of syndromic or nonsyndromic conditions with pituitary tumor predisposition. These include the identification of novel germline variants in patients with familial or simplex pituitary tumors and establishment of novel somatic variants identified through next generation sequencing. Advanced techniques have allowed the exploration of epigenetic mechanisms mediated through DNA methylation, histone modifications and noncoding RNAs, such as microRNA, long noncoding RNAs and circular RNAs. These mechanisms can influence tumor formation, growth, and invasion. While genetic and epigenetic mechanisms often disrupt similar pathways, such as cell cycle regulation, in pituitary tumors there is little overlap between genes altered by germline, somatic, and epigenetic mechanisms. The interplay between these complex mechanisms driving tumorigenesis are best studied in the emerging multiomics studies. Here, we summarize insights from the recent developments in the regulation of pituitary tumorigenesis

Purinergic twelfth receptor 181st position randomized variant functional activity research

Kopsavilkums Darba gaitā tika izvērtēta P2Y12R 181. pozīcijā esošās glutamīnskābes loma receptora mijiedarbībā ar agonistu ADP un antagonistu OSI-10110. P2Y12R varianti ar mutaģenizētu 181. pozīciju tika ekspresēti S. cerevisiae šūnās un aktivēti ar ADP, OSI-10110 un AR-C66096. Tika konstatēts, ka Glu181 aizvietošana ar pēc fizikāli ķīmiskās dabas līdzīgām aminoskābēm, var dažādi ietekmēt receptora un liganda mijiedarbību. Ar QSAR datu analīzi, tika konstatēts, ka P2Y12R Glu181 mijiedarbībā ar ADP un OSI-10110, būtiskas ir aminoskābes hidrofīlās un elektriskās īpašības. Glu181 mijiedarbību ar OSI-10110 nedaudz arī ietekmē aminoskābes fleksibilitāte. OSI-10110 uzrāda zemāku ietekmi uz ADP izraisīto P2Y12R funkcionālo aktivitāti, nekā AR-C66096. Atslēgvārdi: P2Y12R, Saccharomyces cerevisiae, funkcionālā analīze, QSAR.In this study role of glutamic acid at 181th position of P2Y12 on receptor agonist ADP, as well as antagonist OSI-10110 interaction was examined. P2Y12R with replaced 181th amino acid was expressed in S. cerevisiae and activated with ADP, OSI-10110 and AR-C66096. It was found that Glu181 replacement with the physical and chemical nature similar amino acids, can variety affect a receptor and ligand interactions. QSAR data analysis shows that P2Y12R Glu181 interaction with ADP and OSI-10110 are affected by hydrophilic and electrical properties. Glu181 and OSI-181 interaction are affected also by amino acid flexibility. OSI-10110 shows a weaker effect at P2Y12R functional activity, than AR-C66096. Keywords: P2Y12R, Saccharomyces cerevisiae, functional analysis, QSAR

Functional profile of pituitary adenomas and factors affecting it

Adenohipofīzes adenomas ir labdabīgi intrakraniāli endokrīnie audzēji ar potenciāli augstu prevalenci populācijā. Klīniskajā praksē ir attīstītas vairākas adenomu ārstniecības terapijas, taču tās ne vienmēr ir efektīvas. Darbā veikta adenohipofīzes adenomu funkcionalitāti ietekmējošo faktoru raksturošana. Tika noteikti somatotrofajām, laktotrofajām un nehormonālajām adenomām raksturīgi gēnu ekspresijas profili, kā arī funkcionālajiem tipiem nespecifiski somatostatīna un dopamīna receptoru klasteri, kas norāda uz konkrēto receptoru mijiedarbību adenomu darbības ietekmēšanā. Tika konstatēts, ka hormonu sintēzi ierosinošie faktori pozitīvi ietekmē adenomu funkcionālo darbību nomācošo gēnu ekspresiju, tādējādi mazinot hormonu hipersekrēciju un audzēju attīstīšanos. Atslēgvārdi: adenohipofīzes adenomas, gēnu ekspresija, funkcionālais profils.Pituitary adenomas are benign intracranial endocrine tumors with potentially high prevelence in population. In clinical practice, a number of adenomas treatment tactics has been developed, but they are not always effective. In this study we characterise pituitary adenoma functionality affecting factors. We found gene expression profiles specific to somatotroph, lactotroph and non-hormonal pituitary adenomas, and functional types of non-specific somatostatin and dopamine receptor clusters, which seem to differently affect adenomas functionality. It was found that pituitary hormones synthesis-initiating factors positively influence adenomas functional activity depressing protein genes expression, thereby reducing hormone hypersecretion and the risks of tumors development. Keywords: pituitary adenoma, gene expression, functional profile