Genetic and Environmental Factors in Autoimmune Thyroid Disease: Exploring Associations with Selenium Levels and Novel Loci in a Latvian Cohort.

The interplay of genetic, immune and environmental factors strongly contributes to the development of autoimmune thyroid disease (AITD), which can be classified as Graves' disease (GD) or Hashimoto thyroiditis (HT). One of the most studied exogenous factors in the pathogenesis of AITD is selenium, which, in the form of selenoproteins, strengthens the antioxidative defence system of thyroid cells against superoxide production. Furthermore, it modulates inflammatory cytokine release and autoantibody production. The aim of this study was to assess the associations of genetic factors with selenium levels in a cohort of adults with HT and GD and healthy controls from Latvia. A total of 148 GD patients, 102 HT patients and 2442 control participants were included in the study. The genotypes were determined using genome-wide genotyping; imputation was carried out using the TOPMed r2 imputation panel; and association analysis was performed with PLINK v1.90b7. We found three loci associated with GD (LSAMP, HNRNPA3P5, and NTN1) and one locus associated with HT (VAT1L); furthermore, one locus was associated with a serum selenium concentration > 80 µg/L (LINC01544/RNF152/PIGN). The detected associations could be attributed to population-specific effects or unknown stratification in our cohort, and further assessment of these results is required to explain the relationships of genetic traits with AITD and other phenotypes.

Transcriptome of GH-producing pituitary neuroendocrine tumours and models are significantly affected by somatostatin analogues.

Pituitary neuroendocrine tumours (PitNETs) are neoplasms of the pituitary that overproduce hormones or cause unspecific symptoms due to mass effect. Growth hormone overproducing GH-producing PitNETs cause acromegaly leading to connective tissue, metabolic or oncologic disorders. The medical treatment of acromegaly is somatostatin analogues (SSA) in specific cases combined with dopamine agonists (DA), but almost half of patients display partial or full SSA resistance and potential causes of this are unknown. In this study we investigated transcriptomic landscape of GH-producing PitNETs on several levels and functional models-tumour tissue of patients with and without SSA preoperative treatment, tumour derived pituispheres and GH3 cell line incubated with SSA to study effect of medication on gene expression. MGI sequencing platform was used to sequence total RNA from PitNET tissue, pituispheres, mesenchymal stromal stem-like cells (MSC), and GH3 cell cultures, and data were analysed with Salmon-DeSeq2 pipeline. We observed that the GH-producing PitNETs have distinct changes in growth hormone related pathways related to its functional status alongside inner cell signalling, ion transport, cell adhesion and extracellular matrix characteristic patterns. In pituispheres model, treatment regimens (octreotide and cabergoline) affect specific cell proliferation (MKI67) and core functionality pathways (RYR2, COL8A2, HLA-G, ARFGAP1, TGFBR2). In GH3 cells we observed that medication did not have transcriptomic effects similar to preoperative treatment in PitNET tissue or pituisphere model. This study highlights the importance of correct model system selection for cell transcriptomic profiling and data interpretation that could be achieved in future by incorporating NGS methods and detailed cell omics profiling in PitNET model research.

Stress-Related Immune Response and Selenium Status in Autoimmune Thyroid Disease Patients.

Autoimmune thyroid disease (AITD), including Graves' disease (GD) or Hashimoto's thyroiditis (HT), occurs due to genetic susceptibility and environmental factors, among which the role of stressful events remains controversial. This study investigated the relationship between the number and impact of stressful life events in AITD patients with selenium status, and the Th1/Th2/Th17 immune response. The study population included three groups: HT (n = 47), GD (n = 13), and a control group (n = 49). Thyroid function parameters, autoantibody levels, and the plasma levels of cytokines, selenium, selenoprotein P (SeP), and glutathione peroxidase 3 (GPx) activity were measured. Participants filled out the Life Experiences Survey. No significant differences in the number of stressful life events were found among the patients with HT, GD, and the controls. A higher (median (interquartile range)) negative stress level (8 (4-12)) than a positive stress level (3 (1-9)) was found in the HT group. The HT group showed a correlation between SeP and the positive stress level: rs = -0.296, p = 0.048, and the GD group between GPx and the negative stress level (rs = -0.702, p = 0.011). Significant positive correlations between thyroid peroxidase antibody level and the total number of major life events (p = 0.023), the number of major life events in the last 7-12 months, and the number of major life events with no impact and a negative stress level were found. We suggest that the measurements of Th2-related cytokines and selenoproteins could be used as biomarkers for the development of AITD in cases where stress is considered a component cause of the pathogenic mechanism of the disease.

Assessment of Iodine and Selenium Nutritional Status in Women of Reproductive Age in Latvia.

Background and Objectives: Adequate dietary intake of iodine and selenium is essential during pregnancy. While iodine is vital for maternal thyroid function and fetal development, selenium contributes to the regulation of thyroid function and thyroid autoimmunity. This study aimed to assess the consumption of iodine- and selenium-containing products by women of reproductive age and the iodine and selenium nutritional status of pregnant women in Latvia. Materials and Methods: Population health survey (2010-2018) data were used to characterize dietary habits in women of reproductive age. Additionally, 129 pregnant women in the first trimester were recruited; they completed a questionnaire and were tested for thyroid function, urinary iodine concentration (UIC), and serum selenium and selenoprotein P levels. Results: The use of some dietary sources of iodine (e.g., milk and dairy products) and selenium (e.g., bread) has decreased in recent years. Less than 10% of respondents reported the use of iodized salt. The use of supplements has become more common (reported by almost 50% of respondents in 2018). Dietary habits were similar in pregnant women, but the use of supplements was even higher (almost 70%). Nevertheless, most supplements used in pregnancy had insufficient contents of iodine and selenium. Thyroid function was euthyreotic in all women, but 13.9% of participants had a thyroid peroxidase antibodies (TPO-ab) level above 60 IU/mL. The median UIC (IQR) was 147.2 (90.0-248.1) µg/gCr, and 52.8% of pregnant women had a UIC below 150 µg/gCr. The mean selenium (SD) level was 101.5 (35.6) µg/L; 30.1% of women had a selenium level below 80 µg/L. The median selenoprotein P level was 6.9 (3.1-9.0) mg/L. Conclusions: Iodine nutrition in Latvian population of pregnant women was near the lower limit of adequate and a third of the population had a selenium deficiency. Supplements were frequently used, but most did not contain the recommended amounts of iodine and selenium.

Worldwide inertia to the use of cardiorenal protective glucose-lowering drugs (SGLT2i and GLP-1 RA) in high-risk patients with type 2 diabetes.

The disclosure of proven cardiorenal benefits with certain antidiabetic agents was supposed to herald a new era in the management of type 2 diabetes (T2D), especially for the many patients with T2D who are at high risk for cardiovascular and renal events. However, as the evidence in favour of various sodium-glucose transporter-2 inhibitor (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1 RA) accumulates, prescriptions of these agents continue to stagnate, even among eligible, at-risk patients. By contrast, dipeptidyl peptidase-4 inhibitors (DPP-4i) DPP-4i remain more widely used than SGLT2i and GLP-1 RA in these patients, despite a similar cost to SGLT2i and a large body of evidence showing no clear benefit on cardiorenal outcomes. We are a group of diabetologists united by a shared concern that clinical inertia is preventing these patients from receiving life-saving treatments, as well as placing them at greater risk of hospitalisation for heart failure and progression of renal disease. We propose a manifesto for change, in order to increase uptake of SGLT2i and GLP-1 RA in appropriate patients as a matter of urgency, especially those who could be readily switched from an agent without proven cardiorenal benefit. Central to our manifesto is a shift from linear treatment algorithms based on HbA1c target setting to parallel, independent considerations of atherosclerotic cardiovascular disease, heart failure and renal risks, in accordance with newly updated guidelines. Finally, we call upon all colleagues to play their part in implementing our manifesto at a local level, ensuring that patients do not pay a heavy price for continued clinical inertia in T2D.

Case report: recurrent pituitary adenoma has increased load of somatic variants.

BACKGROUND: Pituitary adenomas (PA) have an increased potential for relapse in one to 5 years after resection. In this study, we investigated the genetic differences in genomic DNA of primary and rapidly recurrent tumours in the same patient to explain the causality mechanisms of PA recurrence. CASE PRESENTATION: The patient was a 69-year-old female with non-functional pituitary macroadenoma with extension into the left cavernous sinus (Knosp grade 2) who underwent craniotomy and partial resection in August 2010. Two years later, the patient had prolonged tumour growth with an essential suprasellar extension (Knosp grade 2), and a second craniotomy with partial tumour resection was performed in September 2012. In both tumours, the KI-67 level was below 1.5%. Exome sequencing via semiconductor sequencing of patient germline DNA and somatic DNA from both tumours was performed. Tmap alignment and Platypus variant calling were performed followed by variant filtering and manual review with IGV software. We observed an increased load of missense variants in the recurrent PA tumour when compared to the original tumour. The number of detected variants increased from ten to 26 and potential clonal expansion of four variants was observed. Additionally, targeted SNP analysis revealed five rare missense SNPs with a potential impact on the function of the encoded proteins. CONCLUSIONS: In this case study, an SNP located in HRAS is the most likely candidate inducing rapid PA progression. The relapsed PA tumour had a higher variation load and fast tumour recurrence in this patient could be caused by clonal expansion of the leftover tumour tissue.

Upregulated tissue expression of T helper (Th) 17 pathogenic interleukin (IL)-23 and IL-1ß in Hashimoto's thyroiditis but not in Graves' disease.

T helper (Th) 17 cells and interleukin (IL)-17 play a significant role in the pathogenesis of autoimmune thyroid disease (AITD). However, it has recently become clear that Th17 cells are more heterogeneous and exhibit two different phenotypes, whereas IL-23 and IL-1ß are crucial for the generation of pathogenic Th17 lymphocytes. We aimed to investigate the association between IL-17 and Th17-promoting cytokines in AITD by studying the immunoexpression patterns of IL-17, IL-23, and IL-1ß in thyroid tissue. Following thyroidectomy, 29 patients with AITD (21 cases of Hashimoto's thyroiditis (HT) and 8 cases of Graves' disease (GD)) and 18 patients with colloid goiter, as controls, were enrolled in this study, and immunohistochemistry was performed. The expression level of IL-17 in thyrocytes was significantly higher in HT and GD patients than in colloid goiter patients. Immunopositivity for both IL-23 and IL-1ß was significantly increased in HT patients compared to GD and colloid goiter patients. However, no difference was found between IL-23 or IL-1ß expression in patients with GD and colloid goiter. A positive correlation between IL-17 and IL-23 as well as IL-17 and IL-1ß expression was observed in HT patients (r = 0.574, p = 0.007 and r = 0.461, p = 0.036, respectively). In the GD group, IL-17 was positively correlated with IL-1ß (r = 0.817, p = 0.013) but not with IL-23 expression. We found increased IL-23 and IL-1ß expression in the HT group but not in the GD group. Furthermore, both interleukins were correlated with IL-17 immunopositivity in thyroid tissue, suggesting that pathogenic Th17-promoting cytokines may play a role in HT pathogenesis.

Iodine deficiency during pregnancy: a national cross-sectional survey in Latvia.

OBJECTIVE: Low iodine intake during pregnancy may cause thyroid dysfunction, which results in inadequate fetal brain development. In the absence of a universal salt iodization programme, we conducted a nationwide survey of iodine deficiency in pregnant women in Latvia. DESIGN: A countrywide twenty-cluster survey, with at least twenty women per cluster. Participants completed a questionnaire on dietary habits concerning iodine intake (n 739). Thyroid function (thyroid-stimulating hormone, free thyroxine and thyroperoxidase antibodies) was measured (n 550). Urinary iodine was measured using the ammonium persulfate method (n 696). SETTING: The survey was performed in all regions of Latvia during the spring and autumn seasons in 2013. SUBJECTS: Pregnant women (n 829). RESULTS: The median creatinine (Cr)-standardized urinary iodine concentration (UIC) was 80·8 (interquartile range (IQR) 46·1-130·6) µg/g Cr or 69·4 (IQR 53·9-92·6) µg/l during pregnancy, and 81% of pregnant women had UIC levels below the WHO recommended range of 150-250 µg/g Cr. The UIC was lowest during the first trimester of pregnancy, 56·0 (IQR 36·4-100·6) µg/g Cr, reaching higher concentrations of 87·5 (IQR 46·4-141·7) µg/g Cr and 86·9 (IQR 53·8-140·6) µg/g Cr in the second and third trimesters, respectively. Women taking supplements containing ≥150 µg iodine (6·8% of respondents) had non-significantly higher UIC than did women without supplementation (96·2 v. 80·3 µg/g Cr, respectively, P=NS). Thyroperoxidase antibody concentration did not correlate significantly with UIC: Spearman's ρ=-0·012, P=0·78. CONCLUSIONS: The median UIC indicates iodine deficiency in pregnant women in Latvia. Iodine supplementation (150 µg daily) and regular UIC monitoring should be suggested to overcome iodine deficiency and to reach the recommended levels without inducing autoimmune processes.

Determination of trimethylamine-N-oxide in combination with L-carnitine and γ-butyrobetaine in human plasma by UPLC/MS/MS.

An ultra-high-performance liquid chromatography-mass spectrometry (UPLC/MS/MS) method was developed and validated for the quantification of trimethylamine-N-oxide (TMAO) simultaneously with TMAO-related molecules L-carnitine and γ-butyrobetaine (GBB) in human blood plasma. The separation of analytes was achieved using a Hydrophilic interaction liquid chromatography (HILIC)-type column with ammonium acetate-acetonitrile as the mobile phase. TMAO determination was validated according to valid US Food and Drug Administration guidelines. The developed method was successfully applied to plasma samples from healthy volunteers.

Acute anti-hyperglycaemic effects of an unripe apple preparation containing phlorizin in healthy volunteers: a preliminary study.

BACKGROUND: The health-promoting properties of apples are directly related to the biologically active compounds that they contain, such as polyphenols. The objective of this study was to prepare a low-sugar, fibre- and phlorizin-enriched powder from unripe apples and to gain insight regarding its anti-hyperglycaemic activity in healthy volunteers. RESULTS: The unripe apples (Malus domestica Borkh.) were collected 30 days after the full bloom day; blanched and pressed to obtain apple pomace which was then processed with a food cutter, oven-dried and milled to prepare apple powder. The concentrations of total sugars, water-soluble pectin and phlorizin in the apple preparation were 153.44 ± 2.46, 27.73 ± 0.51 and 12.61 ± 0.15 g kg(-1), respectively. Acute ingestion of the apple preparation improved glucose metabolism in the oral glucose tolerance test (OGTT) in six healthy volunteers by reducing the postprandial glucose response at 15 to 30 min by approximately two-fold (P < 0.05) and by increasing urinary glucose excretion during the 2- to 4-h interval of the OGTT by five-fold (P < 0.05). CONCLUSION: The results obtained indicate that the dried and powdered pomace of unripe apples can be used as a health-promoting natural product for the reduction of postprandial glycaemia and to improve the health of patients with diabetes.

A cross-sectional survey of urinary iodine status in Latvia.

BACKGROUND AND OBJECTIVE: A nationwide survey of schoolchildren was conducted to detect regional differences in urinary iodine excretion in Latvia and to compare the results with data from the newborn thyroid-stimulating hormone (TSH) screening database as well with the results of a similar study performed in Latvia 10 years ago. MATERIALS AND METHODS: We conducted a cross-sectional school-based cluster survey of 915 children aged 9-12 years in 46 randomly selected schools in all regions of Latvia. Urine samples, questionnaires on the consumption of iodized salt and information on socioeconomic status were collected. TSH levels in newborns were also measured. RESULTS: The median creatinine-standardized urinary iodine concentration (UIC) in our study was 107.3µg/g Cr. UIC measurements indicative of mild iodine deficiency were present in 31.6%, moderate deficiency in 11.9% and severe deficiency in 2.8% of the participants. The prevalence of iodine deficiency was the highest in the southeastern region of Latgale and the northeastern region of Vidzeme. The prevalence of TSH values >5mIU/L followed a similar pattern. The self-reported prevalence of regular iodized salt consumption was 10.2%. Children from urban schools had a significantly lower UIC than children from rural schools. CONCLUSIONS: Our findings suggest that although the overall median UIC in Latvian schoolchildren falls within the lower normal range, almost 50% of the schoolchildren are iodine deficient, especially in urban schools and in the eastern part of Latvia. The absence of a mandatory salt iodization program puts a significant number of children and pregnant women at risk.

Solitary fibrous tumor with IGF-II-induced non-islet cell tumor hypoglycemia: a case report and molecular characterization by next-generation sequencing.

Background: Non-islet cell tumor-induced hypoglycemia (NICTH) is a rare, life-threatening medical condition caused by excessive insulin-like growth factor II (IGF-II) secretion from tumors of most commonly mesenchymal origin. Using next-generation sequencing, we have characterized the genome and transcriptome of the resected IGF-II-secreting solitary fibrous tumor from a patient with severe hypoglycemia accompanied by hypoglycemia unawareness. Case presentation: A 69-year-old male patient presenting with abdominal discomfort was examined using computer tomography, revealing a large lesion at the lesser pelvis extending above the umbilicus. As no bone and lymph node metastases were detected, the patient was scheduled for laparotomy. Before surgery, the patient presented with symptoms of severe hypoglycemia. Suppressed C-peptide levels and subsequent hypokalemia indicated a possible case of NICTH. The patient was treated with methylprednisolone (8 mg) to assess hypoglycemia. After the surgery, mild hypoglycemia was present for the postoperative period, and no radiological recurrences were observed 3 and 12 months after discharge. Histopathological examination results were consistent with the diagnosis of malignant solitary fibrous tumor (SFT). Overexpression of IGF-II was confirmed by both immunohistochemistry and RNA sequencing. Further NGS analysis revealed an SFT characteristic alteration-NAB2-STAT6 fusion. Additionally, three deleterious missense variants were detected in oncogenes BIRC6, KIT, and POLQ, and one homozygous in-frame deletion in the RBM10 tumor suppressor gene. Conclusion: While the NAB2-STAT6 fusions are well characterized, the mutational landscape of SFTs remains understudied. This study reports the importance of NGS to characterize SFTs as we detected four coding variants in genes (BIRC6, KIT, POLQ, and RBM10) associated with tumorigenesis that could potentially contribute to the overall pathogenesis of SFT.

Metatranscriptome analysis of blood in healthy individuals and irritable bowel syndrome patients.

Introduction. Although the presence of micro-organisms in the blood of healthy humans is a relatively new concept, there is a growing amount of evidence that blood might have its own microbiome.Gap Statement. Previous research has targeted the taxonomic composition of the blood microbiome using DNA-based sequencing methods, while little information is known about the presence of microbial transcripts obtained from the blood and their relation to conditions connected with increased gut permeability.Aim. To detect potentially alive and active micro-organisms and investigate differences in taxonomic composition between healthy people and patients with irritable bowel syndrome (IBS), we used the metatranscriptomics approach.Methodology. We collected blood samples from 23 IBS patients and 26 volunteers from the general population, and performed RNAseq on the isolated RNA. Reads corresponding to microbial genomes were identified with Kraken 2's standard plus protozoa and fungi database, and re-estimated at genus level with Bracken 2.7. We looked for trends in the taxonomic composition, making a comparison between the IBS and control groups, accounting for other different factors.Results. The dominant genera in the blood microbiome were found to be Cutibacterium, Bradyrhizobium, Escherichia, Pseudomonas, Micrococcus, Delftia, Mediterraneibacter, Staphylococcus, Stutzerimonas and Ralstonia. Some of these are typical environmental bacteria and could partially represent contamination. However, analysis of sequences from the negative controls suggested that some genera which are characteristic of the gut microbiome (Mediterraneibacter, Blautia, Collinsella, Klebsiella, Coprococcus, Dysosmobacter, Anaerostipes, Faecalibacterium, Dorea, Simiaoa, Bifidobacterium, Alistipes, Prevotella, Ruminococcus) are less likely to be a result of contamination. Differential analysis of microbes between groups showed that some taxa associated with the gut microbiome (Blautia, Faecalibacterium, Dorea, Bifidobacterium, Clostridium, Christensenella) are more prevalent in IBS patients compared to the general population. No significant correlations with any other factors were identified.Conclusion. Our findings support the existence of the blood microbiome and suggest the gut and possibly the oral microbiome as its origin, while the skin microbiome is a possible but less certain source. The blood microbiome is likely influenced by states of increased gut permeability such as IBS.

Fasting-Mimicking Diet Reduces Trimethylamine N-Oxide Levels and Improves Serum Biochemical Parameters in Healthy Volunteers.

Elevated plasma levels of trimethylamine N-oxide (TMAO) have been proposed as a diet-derived biomarker of cardiometabolic disease risk. Caloric restriction is the most common dietary intervention used to improve cardiometabolic health; however, novel trends suggest a fasting-mimicking diet (FMD) as a more feasible alternative. FMD is a variation of intermittent fasting, based on caloric restriction and limitation of protein sources of animal origin, applied in daily cycles during a 5-day period. As TMAO is intensively produced by gut microbiota after the consumption of animal-derived products, we aim to investigate whether a 5-day FMD affects plasma TMAO levels and markers of metabolic health. To investigate whether an increase in vegetable intake possesses similar effects on TMAO levels and metabolic parameters, healthy volunteers (n = 24) were subjected to a 5-day FMD and 19 volunteers served as a reference group (VEG). This group of volunteers consumed an additional four servings of vegetables per day, but otherwise stayed on their usual diet. FMD resulted in a twofold decrease in plasma TMAO levels, which was not evident in the volunteers from the VEG group. Moreover, FMD led to a weight loss of 2.8 ± 0.2 kg and a subsequent reduction in BMI compared to baseline. The FMD group exhibited a significant elevation in plasma ketone bodies (14-fold compared to baseline) and a decrease in IGF-1 levels by 37 ± 8 ng/mL. Since fasting glucose and C-peptide levels decreased, all volunteers in the FMD group showed improved insulin sensitivity and a decreased HOMA-IR index. In contrast, in the VEG group, only a slight reduction in plasma levels of fasting glucose and triglycerides was noted. In conclusion, we show that FMD is a viable strategy to reduce plasma levels of TMAO by limiting caloric intake and animal-derived protein consumption. The reduction in the level of TMAO could be an additional benefit of FMD, leading to a reduced risk of cardiometabolic diseases.

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

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.

Whole exome sequencing reveals novel risk genes of pituitary neuroendocrine tumors.

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.

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

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.

Flow cytometric analysis of glyoxalase-1 expression in human leukocytes.

Altered glyoxalase-1 (GLO-1) activity and expression is associated with the development of late diabetic complications, malignancy and oxidative stress- and aging-related diseases. In the present study, we developed a flow cytometry method for GLO-1 detection in human leukocytes isolated from peripheral blood samples to investigate GLO-1 expression in leukocyte subsets from type 1 and 2 diabetes mellitus patients (n = 11) and healthy subjects (n = 8). The flow cytometry analysis of GLO-1 in leukocytes showed that expression index of GLO-1-positive cells was slightly increased in mononuclear leukocytes from diabetic patients. This result correlated with the increase in GLO-1 activity in the whole blood samples of type 2 diabetes patients. In conclusion, the present study demonstrates that flow cytometry is suitable for the detection of the GLO-1 enzyme in human leukocytes and that this method could be used to investigate the fast adaptation of the glyoxalase system related to the pathogenesis of late complications of diabetes mellitus and other glycation stress-related disorders.

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

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.