[Plasma miRNA expression in patients with genetically confirmed multiple endocrine neoplasia type 1 syndrome and its phenocopies].

BACKGROUND: MEN-1 is a rare autosomal dominant disease caused by mutations in MEN1 gene encoding the menin protein. This syndrome is characterized by the occurrence of parathyroid tumors, gastroenteropancreatic neuroendocrine tumors, pituitary adenomas, as well as other endocrine and non-endocrine tumors. If a patient with the MEN-1 phenotype carry no mutations in the MEN1 gene, the condition considers a phenocopy of syndrome (phMEN1). The possible cause of this changes could be changes in epigenetic regulation, particularly in microRNA expression that might affect menin signaling pathways. AIM: to identify differently expressed circulating miRNAs in plasma in patients with genetically confirmed MEN-1 syndrome, its phenocopies and healthy controls. MATERIALS AND METHODS: single-center, case-control study was conducted. We assessed plasma microRNA expression in patients with genetically confirmed MEN-1 (gMEN1), phMEN1 and healthy controls. Morning plasma samples were collected from fasting patients and stored at -80°C. Total RNA isolation was performed using miRNeasy Mini Kit with QIAcube. The libraries were prepared by the QIAseq miRNA Library Kit following the manufacturer. Circulating miRNA sequencing was done on Illumina NextSeq 500 (Illumina). Subsequent data processing was performed using the DESeq2 bioinformatics algorithm. RESULTS: we enrolled 21 consecutive patients with gMEN1 and 11 patients with phMEN1, along with 12 gender matched controls. Median age of gMEN1 was 38,0 [34,0; 41,0]; in phMEN1 - 59,0 [51,0; 60,0]; control - 59,5 [51,5; 62,5]. The gMEN1 group differed in age (p<0.01) but not gender (р=0.739) or BMI (р=0.116) compared to phMEN1 and controls group, the last two groups did not differ by these parameters (p>0.05). 25 microRNA were differently expressed in groups gMEN1 and phMEN1 (21 upregulated microRNAs, 4 - downregulated). Comparison of samples from the phMEN-1 group and relatively healthy controls revealed 10 differently expressed microRNAs: 5 - upregulated; 5 - downregulated. In the gMEN-1 and control groups, 26 differently expressed microRNAs were found: 24 - upregulated; 2 - downregulated. The miRNAs most differing in expression among the groups were selected for further validation by RT-qPCR (in the groups of gMEN1 vs phMEN1 - miR-3613-5p, miR-335-5p, miR-32-5p, miR-425-3p, miR-25-5p, miR-576-5p, miR-215-5p, miR-30a-3p, miR-141-3p, miR-760, miR-501-3p; gMEN1 vs control - miR-1976, miR-144-5p miR-532-3p, miR-375; as well as in phMEN1 vs control - miR-944, miR-191-5p, miR-98-5p). CONCLUSION: In a pilot study, we detected microRNAs that may be expressed differently between patients with gMEN-1 and phMEN-1. The results need to be validated using different measurement method with larger sample size.

[Molecular genetics in diagnosis of Coats disease: combination of oligogenic variants associated with different forms of hereditary retinal dystrophy]. / Molekulyarno-geneticheskie nakhodki pri diagnostike bolezni Koatsa: sochetanie oligolokusnykh izmenenii, svyazannykh s raznymi nozologicheskimi formami nasledstvennoi retinopatii.

Coats disease (OMIM 300216) is a form of hereditary retinal dystrophy, which occurs due to congenital abnormality of retinal vessels and features unilateral exudative vitreoretinopathy. Coats disease mostly occurs sporadically; its genetic cause is still undetermined. Molecular genetic research including whole exome sequencing by the NGS method was used to define a genetic cause of the observed phenotype. Two heterozygous variants in different genomic loci associated with other forms of hereditary retinal dystrophy were detected, a rare variant in the HMCN1 gene c.9571C>T, p.(Arg3191Cys), and a known pathogenic variant in the NPHP4 gene c.2930C>T, p.(Thr977Met). The HMCN1 gene is responsible for dominant age-related macular degeneration (OMIM 603075), pathogenic variants in the NPHP4 gene cause recessive Senior-Løken syndrome 4 (OMIM 266900). These genes encode the proteins that are involved in the regulation of integrity of the blood-retinal barrier in the vascular endothelium (NPHP4) and retinal pigment epithelium (HMCN1). The identified mutation in the NPHP4 gene could lead to decreased function of the NPHP4 protein and contribute to the development of retinal degeneration, potentially of oligogenic nature.

[Differences in plasma miRNA levels in inferior petrosal sinus samples of patients with ACTH-dependent Cushing's syndrome].

BACKGROUND: For the last decades microRNAs (miR) have proven themselves as novel biomarkers for various types of diseases. Identification of specific circulating microRNA panel that differ patient with Cushing's disease (CD) and ectopic ACTH syndrome (EAS) could improve the diagnostic procedure. AIM: to evaluate the differences in miR levels in plasma samples drained from inferior petrosal sinuses in patients with CD and EAS. MATERIALS AND METHODS: single-center, case-control study: we enrolled 24 patients with ACTH-dependent Cushing's syndrome (CS) requiring bilateral inferior petrosal sinus sampling (BIPSS).  Among them 12 subjects were confirmed as CD (males=2, females=10; median age 46,5 [IR 33,8;53,5]) and 12 as EAS (males=4, females=8, median age 54 [IR 38,75;60,75]). BIPSS was performed through a percutaneous bilateral approach. Once catheters were properly placed, blood samples were withdrawn simultaneously from each petrosal sinus and a peripheral vein. Plasma samples from both sinuses were centrifuged and then stored at -80 C. MiRNA isolation from plasma was carried out by an miRneasy Plasma/Serum Kit (Qiagen, Germany) on the automatic QIAcube station according to the manufacturer protocol. To prevent degradation, we added 1 unit of RiboLock Rnase Inhibitor (Thermo Fisher Scientific, USA) per 1 µL of RNA solution. The concentration of total RNA in the aqueous solution was evaluated on a NanoVue Plus spectrophotometer (GE Healthcare, USA). The libraries were prepared by the QIAseq miRNA Library Kit following the manufacturer standard protocols. MiR expression was then analyzed by sequencing on Illumina NextSeq 500 (Illumina, USA). RESULTS: 108 miRNAs were differently expressed (p <0,05) in inferior petrosal sinus samples of patients with CD vs EAS. We divided these miRNAs into 3 groups based on the significance of the results. The first group consisted of samples with the highest levels of detected miR in both groups. Four miRNAs were included: miR-1203 was downregulated in CD vs EAS - 36.74 (p=0,013), and three other were upregulated in CD vs EAS: miR-383-3p 46.36 (p=0,01), miR-4290 6.84 (p=0,036), miR-6717-5p 4.49 (p=0,031). This miRs will be validated in larger cohorts using RT-qPCR. CONCLUSION: Plasma miR levels differ in inferior petrosal samples taken from patients with CD vs EAS. These miRs need to be validated by different methods and in peripheral plasma samples in order to be used as potentially non-invasive biomarkers to differentiate ACTH-dependent CS.

[Expression of plasma microRNA in patients with acromegaly].

BACKGROUND: microRNA is a class of small non-coding RNA molecules involved in posttranscriptional regulation of gene expression. MicroRNAs are detectable in blood in stable concentrations, which makes them promising biomarkers for various diseases. AIM: to assess plasma microRNA expression in patients with active acromegaly compared with healthy controls. MATERIAL AND METHODS: single-center, case-control study: assessment of plasma microRNA in patients with acromegaly compared with healthy controls. Fasting blood samples were drawn and centrifuged at +5С temperature and 3000 rpm for 20 minutes, then aliquoted and frozen at 80C until further analysis. MicroRNA extraction and library preparation was done according to manufacturers instructions. Expression analysis was performed on NextSeq sequencer. Bioinformatic analysis using atropos (adapted deletion), STAR (aligning), FastQC (quality control), seqbuster/seqcluster/miRge2 (microRNA annotation, isomiR and new microRNA search, expression analysis). Primary endpoint of the study differential expression of plasma microRNA in patients with acromegaly compared with healthy controls. RESULTS: we included 12 patients with acromegaly age 33.1 [20; 47], BMI 29.3 kg/m2 [24.0; 39.6], IGF-1 686.1 ng/mL [405.9; 1186.0] and 12 healthy subjects age 36.2 [26; 44], BMI 26.7 kg/m2 [19.5; 42.5], IGF-1 210.4 ng/mL [89.76; 281.90]; gender ratio for both groups 4 males, 8 females. The groups did not differ in gender (p=0.666), age (p=0.551) and BMI (p=0.378). We found decreased expression of four microRNAs in patients with acromegaly: miR-4446-3p 1.317 (p=0.001), miR-215-5p 3.040 (p=0.005), miR-342-5p 1.875 (p=0.013) and miR-191-5p 0.549 (p=0.039). However, none of these changes were statistically significant after adjustment for multiple comparisons (q 0.1). CONCLUSION: we found four microRNAs, which could potentially be downregulated in plasma of patients with acromegaly. The result need to be validated using different measurement method with larger sample size.