The Search for and Functional Analysis of Genetic Variants in microRNA-Binding Sites using Massively Parallel Reporter Assay.

A large-scale search for the genetic variants with a bias in the representation of alleles in transcriptome data (AE SNPs) and the binding sites in microRNA 3'-UTRs was performed and their functional significance was assessed using massively parallel reporter assay (MPRA). Of the 629,559 associated "SNP-gene" pairs (eQTLs) discovered in the human liver tissue according to the GTEx Analysis V8 data, 4394 polymorphic positions in the 3'-UTRs of the genes, which represent the eQTLs for these genes were selected. The TargetScanHuman 7.0 algorithm and PolymiRTS database were searched for the potential microRNA-binding sites. Of the predicted microRNA sites affected by eQTL-SNPs, we selected 51 sites with the best evidence of functionality according to Ago2-CLIP-seq, CLEAR-CLIP, and eCLIP-seq for RNA-binding proteins. For MPRA, a library of the plasmids carrying the main and alternative alleles for each AE SNP (in total, 102 constructs) was created. Allele-specific expression for 6 SNPs was detected by transfection of the HepG2 cell line with the constructed plasmid library and sequencing of target DNA and RNA sequences using the Illumina (MiSeq) platform.

Human-genome single nucleotide polymorphisms affecting transcription factor binding and their role in pathogenesis.

Single nucleotide polymorphisms (SNPs) are the most common type of variation in the human genome. The vast majority of SNPs identified in the human genome do not have any effect on the phenotype; however, some can lead to changes in the function of a gene or the level of its expression. Most SNPs associated with certain traits or pathologies are mapped to regulatory regions of the genome and affect gene expression by changing transcription factor binding sites. In recent decades, substantial effort has been invested in searching for such regulatory SNPs (rSNPs) and understanding the mechanisms by which they lead to phenotypic differences, primarily to individual differences in susceptibility to diseases and in sensitivity to drugs. The development of the NGS (next-generation sequencing) technology has contributed not only to the identification of a huge number of SNPs and to the search for their association (genome-wide association studies, GWASs) with certain diseases or phenotypic manifestations, but also to the development of more productive approaches to their functional annotation. It should be noted that the presence of an association does not allow one to identify a functional, truly disease-associated DNA sequence variant among multiple marker SNPs that are detected due to linkage disequilibrium. Moreover, determination of associations of genetic variants with a disease does not provide information about the functionality of these variants, which is necessary to elucidate the molecular mechanisms of the development of pathology and to design effective methods for its treatment and prevention. In this regard, the functional analysis of SNPs annotated in the GWAS catalog, both at the genome-wide level and at the level of individual SNPs, became especially relevant in recent years. A genome-wide search for potential rSNPs is possible without any prior knowledge of their association with a trait. Thus, mapping expression quantitative trait loci (eQTLs) makes it possible to identify an SNP for which - among transcriptomes of homozygotes and heterozygotes for its various alleles - there are differences in the expression level of certain genes, which can be located at various distances from the SNP. To predict rSNPs, approaches based on searches for allele-specific events in RNA-seq, ChIP-seq, DNase-seq, ATAC-seq, MPRA, and other data are also used. Nonetheless, for a more complete functional annotation of such rSNPs, it is necessary to establish their association with a trait, in particular, with a predisposition to a certain pathology or sensitivity to drugs. Thus, approaches to finding SNPs important for the development of a trait can be categorized into two groups: (1) starting from data on an association of SNPs with a certain trait, (2) starting from the determination of allele-specific changes at the molecular level (in a transcriptome or regulome). Only comprehensive use of strategically different approaches can considerably enrich our knowledge about the role of genetic determinants in the molecular mechanisms of trait formation, including predisposition to multifactorial diseases.

The Experience of Using Multipotent Mesenchymal Stromal Cells in the Treatment of Severe Recurrent Cholangitis in Children with Biliary Atresia after Kasai Surgery.

This article describes the experience of application of multipotent mesenchymal stromal cells in the complex therapy of severe recurrent cholangitis in 2 children with biliary atresia after Kasai surgery. In both children, hepatic cellular insufficiency and portal hypertension developed against the background of long-term inflammatory process poorly controlled by standard therapy, which was the indication for liver transplantation. During the course of mesenchymal stromal cells therapy, the relief of the inflammatory process and functional recovery of the liver were achieved. At the time of preparing the article, the follow-up of two children since the start of multipotent mesenchymal stromal cell therapy was 3 years 9 months and 2 years 6 months. No recurrence of cholangitis was observed in the patients during the follow-up period, the liver function was preserved. There are no indications for liver transplantation at this moment. Thus, despite the fact that the mechanisms of therapeutic action of multipotent mesenchymal stromal cells in biliary atresia require further investigation, we obtained promising results suggesting the possibility of using mesenchymal stromal cells in the treatment of postoperative complications in children with biliary atresia.

[Congenital hypopituitarism with monosomy of chromosome 18].

Congenital hypopituitarism is a rare disease. It can be caused by isolated inborn defects of the pituitary, gene mutations (PROP1, PIT1), and chromosomal abnormalities.Deletions of chromosome 18 (De Grouchy syndrome types 1 and 2) are a group of rare genetic diseases with a frequency of 1:50,000. Hypopituitarism in these syndromes is detected in from 13 to 56% of cases and depends on the size and location of the deleted segment.We have described a series of clinical cases of patients with congenital hypopituitarism due to deletions in chromosome 18. All children had a characteristic dysmorphic features and delayed mental and speech development. Within first months of life, patients developed muscular hypotension, dysphagia, and respiratory disorders. The patients had various congenital malformations in combination with hypopituitarism (isolated growth hormone deficiency and multiple pituitaryhormone deficiencies). In the neonatal period, there were the presence of hypoglycemia in combination with cholestasis.Hormone replacement therapy led to rapid relief of symptoms.Сhromosomal microarray analysis in 2 patients allowed us to identify exact location of deleted area and deleted genes and optimize further management for them.

Mitochondrial DNA maintenance disorders in 102 patients from different parts of Russia: Mutational spectrum and phenotypes.

Currently, pathogenic variants in more than 25 nuclear genes, involved in mtDNA maintenance, are associated with human disorders. mtDNA maintenance disorders manifest with a wide range of phenotypes, from severe infantile-onset forms of myocerebrohepatopathy to late-onset forms of myopathies, chronic progressive external ophthalmoplegia, and parkinsonism. This study represents the results of molecular genetic analysis and phenotypes of 102 probands with mtDNA maintenance disorders. So far, this is the largest Russian cohort for this group of diseases. Mutations were identified in 10 mtDNA maintenance genes: POLG (n = 59), DGUOK (n = 14), TWNK (n = 14), TK2 (n = 8), MPV17 (n = 2), OPA3 (n = 1), FBXL4 (n = 1), RRM2B (n = 1), SUCLG1 (n = 1) and TYMP (n = 1). We review a mutation spectrum for the DGUOK and TWNK genes, that can be specific for the Russian population. In 34 patients we measured the blood mtDNA copy number and showed its significant reduction. Novel variants were found in 41 cases, which significantly expands the mutational landscape of mtDNA maintenance disorders.

rs2072580T>A Polymorphism in the Overlapping Promoter Regions of the SART3 and ISCU Genes Associated with the Risk of Breast Cancer.

We analyzed association of potentially regulatory polymorphisms (rs590352, rs11542583, rs3829202, rs207258, and rs4796672) with breast cancer. A significant association was found between this disease and rs2072580T>A (p=0.001) located in the overlapping promoter regions of the SART3 and ISCU genes. In women with AA and AT genotypes, the risk of breast cancer is higher by 6.7 times (p=0.001) and 12 times (p=0.001), respectively, in comparison with TT genotype. Under a codominant model of inheritance (AT vs AA+TT), the risk of breast cancer was increased by 4.2 times (р=0.001) for the AT genotype. Under a recessive model of inheritance (TT vs AA+TT), the risk of disease was 10-fold higher (р=0.001) for the TT genotype. It has been demonstrated that the T>A substitution affects the binding properties of transcription factors CREB1 and REST.

[Advantages of Kasai procedure through minimally invasive approach in children with biliary atresia (in Russian only)]. / Preimushchestva mini-dostupa pri vypolnenii operatsii Kasai u detei s biliarnoi atreziei.

AIM: To compare early and long-term results of different surgical interventions in children with biliary atresia. MATERIAL AND METHODS: Retrospective analysis included medical records of children with biliary atresia who were treated at the Filatov Munitsipal Children's Hospital and National Medical Research Center for Obstetrics, Gynecology and Perinatology from 2000 to 2018. There were 91 patients. All patients were divided into three groups. Group 1 - conventional Kasai procedure (n=24), group 2 - laparoscopic Kasai surgery (n=45), group 3 - Kasai procedure through minimally invasive approach (n=22). Groups were comparable. RESULTS: Duration of Kasai procedure through minimally invasive approach was 69±12,97 min that was significantly less than in groups 1 and 2 (p1,3=0,006085; p2,3=0,000024). ICU-stay was minimal in group 3 (1.27±0.55 days, p1,3<0,05; p2,3<0,05). Abdominal drainage time was maximal in group 2 (11.28±6.37 days) and minimal in group 3 (5.86±2.39 days, p2,3=0.0002). Early and 2-year postoperative surgical efficiency was similar in all groups. There were no surgical complications in group 3. In group 2 one child had gastrointestinal bleeding followed by successful medication. There were 3 surgical complications in group 3: adhesive intestinal obstruction, small and large intestine perforation and 2 cases of gastrointestinal bleeding. There was one lethal outcome in the first group. Overall annual survival in children with native liver was 81.8%, 2-year - 51.7%. CONCLUSION: Kasai procedure through minimally invasive approach is justified and rational method with certain benefits of open and laparoscopic interventions and can be considered as a method of choice in treatment of children with biliary atresia.

Visceral symptoms as a key diagnostic sign for the early infantile form of Niemann-Pick disease type C in a Russian patient: a case report.

BACKGROUND: Niemann-Pick disease type C is a rare metabolic disease characterized by progressive neurological deterioration with childhood onset, and often results in premature mortality. Niemann-Pick disease type C has an extremely heterogeneous clinical presentation with a wide range of visceral and neurological signs and symptoms that are not specific to the disease, and which progress over varied periods of time. The incidence and epidemiology of Niemann-Pick disease type C in Russia have not been characterized. We report the case of a Russian newborn with early-infantile onset Niemann-Pick disease type C who displayed prolonged neonatal jaundice and hepatosplenomegaly. CASE PRESENTATION: A 5-year-old white boy born to non-consanguineous Russian parents was originally diagnosed with galactosemia at the age of 2 months based on a raised blood galactose level. A galactose-free and lactose-free diet resulted in achievement of a normal galactose level, but hepatosplenomegaly and cholestatic signs persisted. Liver biopsy results hinted at possible Niemann-Pick disease type C, but differential diagnostic investigations for progressive familial intrahepatic cholestasis type 2 (Byler syndrome) indicated a heterozygous genotype suggestive of this disease. Further, progressive neurological symptoms prompted additional genetic analyses for possible Niemann-Pick disease type C, from which an as-yet unreported combination of known NPC1 gene mutations was identified, and a final diagnosis of Niemann-Pick disease type C was established. The patient subsequently developed typical neurological symptoms of early-infantile Niemann-Pick disease type C, including vertical supranuclear ophthalmoparesis and cerebellar ataxia. Miglustat therapy was initiated 2.5 years ago, and some improvements in movement and speech have since been observed. CONCLUSIONS: This case illustrates the continued challenges associated with diagnosing Niemann-Pick disease type C based on the appearance of nonspecific cholestatic symptoms. Based on this case we recommend examination of all newborns and children who display unexplained cholestasis or isolated splenomegaly/hepatosplenomegaly during the first months of life for other signs of possible Niemann-Pick disease type C.

[Determination of glutamic acid in biological material by capillary electrophoresis].

The conditions for the identification and determination of Glutamic acid by capillary zone electrophoresis without their preliminary derivatization have been optimized. The effect of concentration of buffer electrolyte and pH on determination of Glutamic acid has been investigated. It is shown that the 5 Mm borate buffer concentration and a pH 9.15 are optimal. Quantitative determination of glutamic acid has been carried out using a linear dependence between the concentration of the analyte and the area of the peak. The accuracy and reproducibility of the determination are confirmed by the method "introduced - found". Glutamic acid has been determined in the placenta homogenate. The duration of analysis doesn't exceed 30 minutes. The results showed a decrease in the level of glutamic acid in cases of pregnancy complicated by placental insufficiency compared with the physiological, and this fact allows to consider the level of glutamic acid as a possible marker of complicated pregnancy.