Analysis of mutations spectrum in the ATP7B gene in patients with Wilson disease using massively parallel sequencing.

The study aimed to search for mutations in the ATP7B gene using massively parallel sequencing in patients with Wilson disease in the Tomsk region. For 42 patients with suspected Wilson's disease (aged from 1 to 33 years) was performed molecular genetic analysis. Enrichment of the interest genome regions was carried out by the long-range PCR. DNA libraries with ligated adapters were constructed with Nextera DNA Flex (Illumina, USA) kit. Sequencing was performed on the Illumina MiSeq platform (Illumina, USA). As a result of this work, we identified 9 pathogenic genetic variants. All variants were previously described in the literature and were found in patients with Wilson's disease. Five missense mutations, one splice site mutation, and 3 frameshift mutations were identified. In patients with Wilson's disease in the Tomsk region, the most common variant was c.3207C>A, this variant is the most common both in the Russian Federation and in other European populations. Also, a pathogenic variant c.3036dupC was found, which is probably endemic to the Russian Federation.

Morphological and molecular analysis of rose cultivars from the Grandiflora and Kordesii garden groups.

The breeding of remontant rose cultivars that are resistant to diseases and adverse conditions, with high decorative value and continuous flowering is the most important task during work with the gene pool of garden roses. Currently, intercultivar hybridization within a single garden group has largely outlived its usefulness. It is necessary to breed for highly decorative forms or cultivars that have outstanding resistance, morphological characters and patterns of seasonal rhythms, and use these plants as parental forms in further breeding. This study represents a comparative analysis of rose cultivars from two garden groups, Grandiflora (Gurzuf, Lezginka, Korallovy Syurpriz, Queen Elizabeth, Komsomolsky Ogonyok, Love) and Rosa Kordesii (Letniye Zvyozdy, Dortmund, Gutsulochka). These cultivars proved themselves during many years of testing in harsh climatic conditions. The objectives of the study were to determine the genetic relationship within the groups and to assign phenotypically different cultivars to one or another garden group. The analysis was carried out by morphological, phenological and ISSR markers. According to the phenological observations on the Grandiflora cultivars, Komsomolsky Ogonyok had later budding and flowering stages. Polymorphic data generated from the ISSR markers showed that this cultivar was the most distant from the others and formed a separate cluster on the dendrogram. A comparison of the morphological characters (flower diameter, number of petals, peduncle length, bush height) showed a significant difference ( p < 0.05) between Komsomolsky Ogonyok and the other Grandiflora cultivars. A dendrogram based on a molecular analysis showed a lack of close relationships between Komsomolsky Ogonyok and the Kordesii group, which formed a separate cluster. A pairwise comparison of the morphological characters in Komsomolsky Ogonyok with the Kordesii group revealed a significant ( p <0.05) difference in three of the four characters studied. The exceptions were flower diameter when comparing with Dortmund and Letniye Zvyozdy and peduncle length when comparing with Gutsulochka. Although Komsomolsky Ogonyok has a pattern of seasonal development similar to Dortmund in the Kordesii group, the molecular analysis did not assign the former to this group of roses. The cultivars that have valuable characters that no average rose does and that are phenotypically different from such roses represent the most valuable breeding material.

Aberrant methylation of placental development genes in chorionic villi of spontaneous abortions with trisomy 16.

In humans, aneuploidy is incompatible with the birth of healthy children and mainly leads to the death of embryos in the early stages of development in the first trimester of pregnancy. Trisomy 16 is the most common aneuploidy among spontaneous abortions of the first trimester of pregnancy. However, the mechanisms leading to the death of embryos with trisomy 16 remain insufficiently investigated. One of these potential mechanisms is abnormal placental development, including aberrant remodeling of spiral arteries. Spiral artery remodeling involves the migration of trophoblast cells into the maternal spiral arteries, replacing their endothelium and remodeling to ensure a stable embryonic nutrition and oxygen supply. This is a complex process which depends on many factors from both the embryo and the mother. We analyzed the methylation level of seven genes (ADORA2B, NPR3, PRDM1, PSG2, PHTLH, SV2C, and TICAM2) involved in placental development in the chorionic villi of spontaneous abortions with trisomy 16 (n = 14), compared with spontaneous abortions with a normal karyotype (n = 31) and the control group of induced abortions (n = 10). To obtain sequencing libraries, targeted amplification of individual gene regions using designed oligonucleotide primers for bisulfite-converted DNA was used. The analysis was carried out using targeted bisulfite massive parallel sequencing. In the group of spontaneous abortions with trisomy 16, the level of methylation of the PRDM1 and PSG2 genes was significantly increased compared to induced abortions (p = 0.0004 and p = 0.0015, respectively). In the group of spontaneous abortions, there was no increase in the level of methylation of the PRDM1 and PSG2 genes, but the level of methylation of the ADORA2B gene was significantly increased compared to the induced abortions (p = 0.032). The results obtained indicate the potential mechanisms of the pathogenetic effect of trisomy 16 on the placental development with the participation of the studied genes.

Specific shoot formation in Miscanthus saccharif lorus (Poaceae) under different environmental factors and DNA passportization using ISSR markers.

The generic complex Miscanthus Anderss. (Poaceae) is a unique example among herbaceous plants characterized by high values of growth of aboveground vegetative mass and practical use as a valuable source of alternative energy. Miscanthus is one of the most eff icient solar energy accumulators, and since phytomeliorative use implies the cultivation of these resource plants in inconvenient and semi-shady areas, the question about the effect of insuff icient lighting on the productivity of Miscanthus arises. As a result of a long-lasing introduction effort, the Central Siberian Botanical Garden SB RAS created a population of Miscanthus saccharif lorus (Maxim.) Benth., which has good prospects for growing under the conditions of the forest-steppe area in Western Siberia. The goals of our study were: (1) to determine the peculiarities of shoot formation, (2) to assess the cellulose and lignin accumulation in M. saccharif lorus populations under different lighting conditions and (3) to perform a DNA passportization of the Miscanthus population by ISSR marking. Evaluation of shoot formation and the amount of accumulated cellulose and lignin in plants was carried out under different degrees of illumination: one variant was grown in a sunny area, and the other, in partial shade. As a result of analysis of variance, it was found that the number of shoots does not depend on environmental conditions, but on the age of the plant, while environmental conditions have a signif icant effect on plant height. Although the samples of both M. saccharif lorus variants were characterized by different rates of creation of a continuous projective cover, plants in semi-shaded areas formed up to 89.34 % of shoots compared to their peers in illuminated areas, which did not affect signif icantly the size of the aboveground mass and the cellulose content in it. As a result of ISSR-analysis of genomic DNA in the M. saccharif lorus population, unique molecular polymorphic fragments were identif ied, which can be used for identif ication and DNA passportization at the inter-population level. Thus, the complex use of M. saccharif lorus as a valuable meliorative and bioenergetic culture is due to the high adaptive potential of this species. It was found that the illumination factor has virtually no effect on the amount of the cellulose content in the shoot, and a reduced content of the technologically undesirable lignin was observed in plants growing in the partial shade conditions.

Generation of an induced pluripotent stem cell line ICGi030-A from a Wilson's disease patient carrying a frameshift mutation p.Lys1013fs and missense mutation p.H1069Q in the ATP7B gene.

Wilson's disease is a rare autosomal recessive disorder of copper metabolism. The copper accumulation in the viscera appears due to the functional impairment of copper-transporting ATPase, which is encoded by the ATP7B gene. In this study, PBMCs of a patient with two ATP7B mutations were reprogrammed. The first mutation is a missense mutation p.H1069Q, which is the most frequent mutation in the human population. At the same time, the second one is a frameshift mutation p.Lys1013fs. The generated iPSC line had a normal karyotype, maintained the original genotype, expressed pluripotency markers, and demonstrated the ability to differentiate into derivatives of the three germ layers.

Generation of two induced pluripotent stem cell lines from peripheral blood mononuclear cells of a patient with Wilson's disease.

Wilson's disease is an inherited disorder associated with copper accumulation in the liver, brain and other vital organs. Wilson's disease is caused by mutations in the ATP7B gene. Over 300 mutations of ATP7B have been described. Despite the disease is autosomal recessive, the patient whose PBMCs were reprogrammed in the study harbours heterozygous mutation c.3207C > A (p.H1069Q). Detailed analysis of the ATP7B complete gene sequencing data has not revealed other known disease associated mutation. The generated iPSC lines maintained the original genotype, expressed pluripotency markers, had normal karyotype and demonstrated the ability to differentiate into derivatives of the three germ layers.