The molecular complexity of COL2A1 splicing variants and their significance in phenotype severity.

Pathogenic single nucleotide variants (SNVs) found in the COL2A1 gene are associated with a broad range of skeletal dysplasias due to their impact on the structure and function of the Col2a1 protein. However, the molecular mechanisms of some nucleotide variants detected during diagnostic testing remain unclear. The interpretation of missense and splicing variants caused by SNVs poses a significant challenge for clinicians. In this work, we analyzed 22 splicing variants in the COL2A1 gene which have been found in patients with COL2A1-associated skeletal dysplasias. Using a minigene system, we investigated the impact of these SNVs on splicing and gained insights into their molecular mechanisms and genotype-phenotype correlations for each patient. The results of our study are very useful for improving the accuracy of diagnosis and the management of patients with skeletal dysplasias caused by SNVs in the COL2A1 gene.

Generation of two iPSC lines from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12.

We generated two human induced pluripotency stem cell (hiPSC) lines, RCMGi011-A and 11-B, from skin fibroblast from patient with Mucopolysaccharidosis IV B type and autosomal recessive non-syndromic hearing loss 12 using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit. We verified variant c.808 T > G and insertion in GLB1 gene, as well as two mutations, c.6992 T > C and c.805C > T, in CDH23 gene which lead to autosomal recessive hearing loss type 12. We have demonstrated normal karyotype of hiPSCs and capacity for cell differentiation into three germ layers.

Digital Technologies for Monitoring and Forecasting the Environmental Situation in Siberia.

This article is dedicated to ecological monitoring of Siberia. The regional features of environmental problems are presented, and an integrated approach to the organization of digital monitoring as a tool for understanding what is happening and its forecasting is proposed. An approach to the digitalization of environmental monitoring based on digital platforms, which provide the collection, storage, and processing of large amounts of data in a distributed network of sensors, and satellite information, as well as services for modeling and forecasting, is considered. The results of digital monitoring in the Baikal natural territory are presented. This article is based on a report heard at the RAS Presidium meeting on June 22, 2021.

The Melatonin Receptor CAND2/PMTR1 Is Involved in the Regulation of Mitochondrial Gene Expression under Photooxidative Stress.

Melatonin is a signaling molecule that mediates multiple stress-dependent reactions. Under photooxidative stress conditions generating intensive ROS production, exogenous melatonin (50 µM) contributed to maintaining the expression of mitochondrial encoded genes and up-regulation of RNA-polymerase genes RPOTm and RPOTmp, operating through the CAND2 receptor and α-subunit of the heterotrimeric G protein GPA1 coupled with CAND2. Unlike wild-type plants, mutants with defective CAND2 and GPA1 genes exhibited no decrease in the alternative pathway of leaf respiration, as well as the activity of an alternative oxidase, and the expression of the AOX1a gene. At the same time, the protective effect of exogenous melatonin on some physiological indicators did not depend on the receptor and was associated with the direct antioxidant function of the regulator. Thus, melatonin under photooxidative stress conditions can act as an antioxidant and as a hormone capable of regulating the expression of nuclear and organelle genes through the components of melatonin signal perception.

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.

Cold Stress Activates the Expression of Genes of the Chloroplast Transcription Apparatus in Arabidopsis thaliana Plants.

The physiological and molecular responses of Arabidopsis thaliana plants to cold stress were studied. Exposure to a low non-freezing temperature (4°C, 5 days) caused a decrease in the physiological functions and activity of a number of photosynthetic genes and elevation in expression of the cold stress gene COR15a, the product of which protects chloroplasts. It was shown for the first time that in parallel to a general inhibition of physiological functions under hypothermia, an increase in the expression of most genes for the chloroplast transcription apparatus was observed. This is obviously one of the compensatory mechanisms of adaptation aimed to maintain cellular homeostasis and physiological functions under hypothermia.

Screening of West Siberian patients with primary congenital glaucoma for CYP1B1 gene mutations.

Primary congenital glaucoma (PСG) is a visual organ pathology that leads to progressive blindness and poor vision in children. Its main cause is an anomaly of the anterior chamber angle. Most cases of PСG are sporadic, but familial cases with an autosomal recessive (predominantly) and autosomal dominant (rare) type of inheritance have been described. Congenital glaucoma is a rare condition (1 case per 10,000-20,000 newborns), but its prevalence is substantially higher (up to 1 case per 250 newborns) in countries where consanguineous marriages are common. Mutations in the CYP1B1 gene, which encodes cytochrome P450 1B1, are the most common cause of autosomal recessive primary congenital glaucoma. This enzyme is known to be involved in retinoic acid metabolism and is necessary for normal eye development. The aim of this work was to assess the polymorphism of the CYP1B1 gene among West Siberian patients with primary congenital glaucoma. Direct automatic Sanger sequencing of exons and adjacent splicing sites of the CYP1B1 gene was carried out in 28 people with the PCG phenotype from a West Siberian region. As a result, in the sample of the white population we examined, pathogenic variants previously described in other ethnic groups were revealed: E387K (rs55989760), R444* (rs377049098), R444Q (rs72549376), and P437L (rs56175199), as well as novel single-nucleotide deletion p.F114Lfs*38 in the CYP1B1 gene. The latter can cause a frame shift, changed amino acid composition, and a formation of truncated in the protein. None of the detected mutations were found in the control sample of ophthalmologically examined individuals without PCG (100 people). Variants R444* (rs377049098) and R444Q (rs72549376) were not found in the general population sample either (576 randomly selected West Siberia residents). All the detected mutations caused the development of the autosomal recessive form of primary congenital glaucoma. The most severe clinical phenotype was observed in carriers of mutations in codon 444 of the gene. Consequently, in children with signs of increased intraocular pressure, molecular genetic analysis of the CYP1B1 gene is advisable for early diagnosis and timely initiation of PCG therapy.

α-Galactosidase A/lysoGb3 ratio as a potential marker for Fabry disease in females.

Fabry disease (FD [MIM:301500]) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene. Deficient activity of its product, lysosomal enzyme α-galactosidase A (α-Gal A), leads to excessive accumulation of glycosphingolipids in cells of multiple organs. The establishing of the diagnosis is challenge in female patients because of milder clinical manifestation and normal α-Gal A activity. The globotriaosylsphingosine (lysoGb3) is described as a more sensitive diagnostic biomarker for females with pathogenic mutation in the GLA gene. Thus, the aim of this study is to improve the biochemical diagnostic efficiency for FD in females. Here we report the α-Gal A/lysoGb3 ratio as the novel biochemical criteria for diagnosis of female patients with FD, using dried blood spots (DBS) as test samples. It showed 100% sensitivity in distinguishing our group of 35 female patients from control (n = 140). Whereas measurement of α-Gal A and lysoGb3 alone showed 8.6% and 74.4% respectively. A new approach of using the ratio of α-Gal A activity to lysoGb3 concentration in DBS may provide a more accurate screening tool for identification of FD females.

Cytokinins and Abscisic Acid Regulate the Expression of the Genes for Plastid Transcription Apparatus during Heat Shock.

The treatment of Arabidopsis thaliana plants with exogenous cytokinin (CK) followed by heat shock (HS) activated the expression of the genes for the plastid transcription machinery but adversely affected the plant viability. Abscisic acid (ABA), conversely, promoted maintaining the resistance to HS and had differentially affected different components of the plastid transcriptional complex. This hormone suppressed the accumulation of transcripts of PEP genes and the genes encoding PAP proteins, which are involved in DNA-RNA metabolism. However, it had no effect or activated the expression of NEP genes and PAP genes, which are involved in the redox regulation, as well as the genes encoding the stress-inducible trans-factor (SIG5) and the plastid transcription Ser/Thr protein kinase (cpCK2). Thus, for the adaptation of plants to elevated temperatures, both increase and decrease in the expression of the genes for the plastid transcriptional machinery with the involvement of various regulatory systems, including phytohormones, are equally significant.

The novel synonymous variant in LIPA gene affects splicing and causes lysosomal acid lipase deficiency.

Lysosomal acid lipase deficiency (LALD; MIM#278000) is a continuum of autosomal recessive diseases caused by defects in the gene LIPA and historically divided into two phenotypes: severe infantile-onset form called Wolman disease (WD) and childhood/adult-onset form known as cholesteryl ester storage disease (CESD). We report a novel synonymous homozygous variant c.600G > A in LIPA of a patient with LALD. Functional analysis of the patient cDNA and minigene assay revealed this variant as the cause of exonic cryptic splice site activation and 63 b.p. deletion in exon 6. To investigate the impact of this in-frame deletion on protein function, we performed 3D modeling of the human lysosomal acid lipase and showed the alteration of highly conservative region in close proximity to protein active site, which may completely eliminate the enzymatic activity. Using transcript specific real-time quantitative PCR method, we evaluated the relative ratio of the patient's wild type transcript isoform which is significantly reduced and correlates with severe childhood-onset variant of LALD.

The Unique Spectrum of Mutations in Patients with Hereditary Tyrosinemia Type 1 in Different Regions of the Russian Federation.

BACKGROUND: Hereditary tyrosinemia (HT1) is an autosomal recessive disorder characterized by impaired tyrosine catabolism because of fumarylacetoacetate hydrolase deficiency. HT1 is caused by homozygous or compound heterozygous mutations in the FAH gene. The HT1 frequency worldwide is 1:100,000-1:120,000 live births. The frequency of HT1 in the Russian Federation is unknown. AIM: To estimate the spectrum of mutations in HT1 in several ethnic groups of the Russian Federation. MATERIALS AND METHODS: From 2004 to 2017, 43 patients were diagnosed with HT1. The analysis of amino acids and succinylacetone was performed using NeoGram Amino Acids and Acylcarnitines Tandem Mass Spectrometry Kit and a Sciex QTrap 3200 quadrupole tandem mass spectrometer. Bi-directional DNA sequence analysis was performed on PCR products using an ABI Prism 3500. RESULTS: In the Russian Federation, the most common mutation associated with HT1 (32.5% of all mutant alleles) is c.1025C>T (p.Pro342Leu), which is typical for the Chechen ethnic group. Patients of the Yakut, the Buryat, and the Nenets origins had a homozygous mutation c.1090G>C (p.Glu364Gln). High frequency of these ethnicity-specific mutations is most likely due to the founder effect. In patients from Central Russia, the splicing site mutations c.554-1G>T and c.1062+5G>A were the most prevalent, which is similar to the data obtained in the Eastern and Central Europe countries. CONCLUSION: There are ethnic specificities in the spectrum of mutations in the FAH gene in HT1. The Chechen Republic has one of the highest prevalence of HT1 in the world.

[Spectrum of pathogenic mtDNA mutations in Leber hereditary optic neuropathy families from Siberia].

The results of clinical, genealogical and molecular investigation of eighteen families with Leber hereditary optic neuropathy (LHON), identified on the territory of Siberia during the period from 1997 to 2005, are presented. Comprehensive analysis of mitochondrial genome variations in probands and their matrilineal relatives revealed the presence of relatively frequent (G11778A, G3460A, and T14484C), as well as rare and new mutations with the established or presumptive pathological effect (T10663C, G363A, C4640T, and A14619G). The G11778A mutation was detected in nine pedigrees (50%), mostly in the families of ethnic Russians. In eight of these families G11778A was found in preferred association with the coding-region substitutions, typical of western Eurasian mtDNA lineage (haplogroup) TJ. On the contrary, the G3460A mutation was detected in the three families belonging to the indigenous Siberian populations (Tuvinians, Altaians, and Buryats). It was associated with clearly different haplotypes of eastern Eurasian haplogroups, C3, D5, and D8. Unexpectedly, the G3460A de novo mutation was found in a large Tuvinian pedigree. At the same time, in eleven out of fourteen families of Caucasoid origin pathogenic mutations in the ND genes were associated with the T4216C and C1542A coding-region mutations, marking the root motif of haplogoup TJ. It is suggested that phylogenetically ancient mutations could have provided their carriers with the adaptive advantages upon the development of Central and Northern Europe at the end of the last glaciation (10 000 to 9 000 years ago), thereby, contributing to the preservation of weekly pathogenic LHON mutations, appearing at specific genetic background.

[The mitochondrial genome and human mitochondrial diseases]. / Mitokhondrial'nyi genom i mitokhondrial'nye bolezni cheloveka.

To date, more than 100 point mutations and several hundreds of structural rearrangements of mitochondrial DNA (mtDNA) are known too be connected with characteristic neuromuscular and other mitochondrial syndromes varying form those causing death at the neonatal stage to diseases with late ages of onset. The immediate cause of mitochondrial disorders is a defective oxidative phosphorylation. Wide phenotypic variation and the heteroplasmy phenomenon, which some authors include in mutation load, are characteristic of human mitochondrial diseases. As the numbers of cases identified and pedigrees described increase, data on the genotype--phenotype interaction and the structure and frequency of pathogenic and conditionally pathogenic mtDNA mutations in human populations are rapidly accumulated. The data on the genetics and epidemiology of mitochondrial diseases are not only important for differential diagnosis and genetic counseling. Since both neutral and mildly pathogenic mutations of mtDNA are progressively accumulated in maternal phyletic lines, molecular analysis of these mutations permits not only reconstruction of the genealogical tree of modern humans, but also estimation of the role that these mutations play in natural selection.

[Evolution of subpopulations of cells of transplantable tumors following x-ray irradiation]. / Evoliutsiia subpopuliatsii kletok perevivaemykh opukholei posle rentgenovskogo oblucheniia.

The paper is devoted to a study of the structure of subpopulations of 3 experimental tumors (ascitic Ehrlich carcinoma, melanoma B-16 and Lewis lung adenocarcinoma) in their progression and following single local X-radiation. At the same time some functional indices of cell viability were investigated over time: intracellular pH, intensity of respiration, the level of cell protein, the level of lipid peroxidation (according to the content of one of its end-products--malonic dialdehyde). Considerable differences of subpopulations within one tumor and between different tumor types were established. The relationship of functional differences of these subpopulations with the clonogenicity of forming cells was shown. X-radiation caused inhomogeneous changes in the structure of subpopulations, the above indices changed. The authors have analyzed the regularity of these changes, their relationship with radiosensitivity or radioresistance of tumor cell subpopulations.

[Potentiation of the selective antitumoral effect of cyclophosphamide and 5-fluorouracil by its combination with amphotericin B and diacarb]. / Povyshenie izbiratel'nosti protivoopukholevogo deistviia tsiklofosfana i 5-ftoruratsila pri ikh sochetanii s amfoteritsinom B i diakarbom.

The paper deals with the results of experiments which were carried out on 10 tumor models and used approximately 1,000 animals (mice and rats of different strains). Such cytostatic drugs as cyclophosphamide (alkylating agent) and 5-fluorouracil (antimetabolite) showed a significantly enhanced antitumor and metastation--preventing effect when used in combination with polyene antibiotic amphotericin B and a diuretic drug--diacarb. This was matched by a pronounced amelioration of toxic side effects and an increase in the animals' lifespan. The said method of treatment should be integrated with oncological practice.

[Effect of amphotericin B on the diuretic action of diacarb and various indicators of water-salt metabolism in healthy and tumor-bearing animals]. / Vliianie amfoteritsina B na diureticheskii éffekt diakarba i nekotorye pokazateli vodno-solevogo obmena u zdorovykh zhivotnykh i zhivotnykh s opukholiami.

The experiments with albino rats, Wistar rats and SHK mice showed that the combined use of amphotericin B and diacarb markedly potentiated the effect of the latter: the amount of the urine excreted within 6 h and the renal excretion of sodium ions increased 1.8 and 1.5 times, respectively. Analogous trends were observed in the experiments with stabilization of the animal water balance by 2 per cent water loading. The studies on rats with Pliss lymphosarcoma showed that the diuresis level in the tumor-bearing animals was 3 times lower than that in healthy rats. The treatment with 5-fluorouracil lowered this index more significantly. With the use of amphotericin B, diacarb and their combination the urination level came to normal. The cyclophosphamide therapy of mice with sarcoma 180 was accompanied by accumulation of water in the tumor tissue. The combined use of the cytostatic with the polyenic antibiotic and diuretic resulted in a significant lowering of this index. In rats with Walker-256 carcinosarcoma, the treatment with the above three drugs lowered the sodium ion concentration and increased the calcium ion concentration in the tumor tissue. At the same time the electrolyte level in some organs not affected with malignant tumor (spleen, thymus, liver and adrenal glands) returned to normal.