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.

Novel intronic variant in PALB2 gene and effective prevention of Fanconi anemia in family.

Despite the acceptance of NextGen sequencing as a diagnostic modality suitable for probands and carriers of Mendelian diseases, its efficiency in identifying causal mutations is limited by both technical aspects of variant call algorithms and by imperfect, consensus-based criteria for assessing the pathogenicity of the findings. Here we describe the medical history of the family with a child born with Fanconi anemia. In this case, typical diagnostic routines were complicated by unusual combination of mutations. PALB2 variant NM_024675.3:c.172_175delTTGT (p.Gln60Argfs) in maternal sample, previously classified as a definitely pathogenic frameshift mutation, was in compound heterozygous state with PALB2 NM_024675.3:c.3114-16_3114-11del (p.Asn1039Glyfs*7), which led to validated PALB2 exon 11 skipping event in paternal locus. Findings enabled the development of the PGТ and successful selection of two mutation-free embryos. We show that even in absence of definitive exome findings, clinician-guided research inquiries into the structure and function of the suspected loci allow definitive diagnosis. Described case provides an example of a crucial input of an investigational workflow in genetic prognosis and successful PGT.

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.

[Efficiency of the miRNA- mRNA Interaction Prediction Programs].

miRNAs play a key role in regulation of gene expression. Nowadays it is known more than 2500 human miRNAs, while a majority of miRNA-mRNA interactions remains unidentified. The recent development of a high-throughput CLASH (crosslinking, ligation and sequencing of hybrids) technique for discerning miRNA-mRNA interactions allowed an experimental analysis of the human miRNA-mRNA interactome. Therefore, it allowed us, for the first time, make an experimental analysis of the human miRNA-mRNA interactome as a whole and an evaluation of the quality of most commonly used miRNA prediction tools (TargetScan, PicTar, PITA, RNA22 and miRanda). To estimate efficiency of the miRNA-mRNA prediction tools, we used next parameters: sensitivity, positive predicted value, predictions in different mRNA regions (3' UTR, CDS, 5' UTR), predictions for different types of interactions (5 classes), predictions of "canonical" and "nocanonical" interactions, similarity with the random generated data. The analysis revealed low efficiency of all prediction programs in comparison with the CLASH data in terms of the all examined parameters.

Isolation and Characterization of Human Myoblast Culture In Vitro for Technologies of Cell and Gene Therapy of Skeletal Muscle Pathologies.

We analyzed cultures of 5 independent myoblast lines from human skeletal muscles. It was shown that the content of desmin-positive cells in cultures at early passages exceeds 90%. Typical morphofunctional signs of myogenic differentiation disturbances were identified and their dynamics was studied. Signs of alternative adipogenic and chondrogenic differentiation of cells were revealed. Based on these data, limitations for the use of myoblast cultures of certain passages for biomedical research and cell therapy were evaluated.

[Clinical and genetic characteristics of facioscapulohumeral muscular dystrophy Landuzi-Dezherina type 1]. / Kliniko-geneticheskie kharakteristiki litse-plechelopatochnoi miodistrofii Landuzi-Dezherina tipa 1.

AIM: To describe clinical and genetic characteristics of patients from the Russian population with a variety of phenotypic variants of facioscapulohumeral muscular dystrophy Landuzi-Dezherina type 1 (FSHD 1). MATERIAL AND METHODS: The material for the study were blood samples of 16 patients from 15 unrelated families residing in the territory of the Russian Federation, between the ages of 6 to 66 years, with symptoms of FSHD. Diagnosis was based on genealogical data analysis, neurological examination, electroneuromyographic study, indicators of activity of creatine phosphokinase (CPK) in the blood serum and molecular genetic analysis of the results, aimed at the analysis of macrosatellite D4Z4 repeats on chromosome 4. RESULTS AND CONCLUSION: The study established the diagnosis of FSHD1 in 75% of patients. The correlation of the severity and phenotypic spectrum of FSHD1 with the age of onset was found. There was the significant clinical heterogeneity even among the 1st degree relatives in the same family. The correlation between macrosatellite D4Z4 repeats and clinical features of FSHD1 described previously in the literature was not observed.

[The Mechanisms of Transgenerational Inheritance and Their Potential Contribution to Human Phenotypes].

As of today, classical genetics has already completed the majority of groundwork to describe the laws of inheritance, identify the causes of many human diseases, and dissect the mechanisms of transfer of genetic information from parents to offspring. However, recent studies indicate that inheritance of phenotypic traits may also occur through nongenetic factors, in particular, through epigenetic factors, that manifest their effects in a transgenerational fashion. This review discusses findings in the area of transgenerational inheritance that open a new era in modern genetics. We discuss the mechanisms of transgenerational inheritance, including DNA methylation, histone modifications, and noncoding RNA transfer, and give an overview of the approaches to detect transgenerational effects in humans.

[Cloning of alternative isoforms of the catalytic subunit of the human thelomerase (htert)].

Most human tumors, including cervical cancer, are characterized by telomerase activation (cell proliferation activation enzyme). Such activation is implemented in the elongation of the terminal segments (telomeres) of the telomerase chromosome. The gene of the enzyme is RNA-encoded, the RNA in tumors being observed in a few isoforms. The hTERT RNA role in cell activation and control was simulated using cervical cancer, as well as its pretumoral states (CIN), as a model object. The goal of this work was to clone of the human hTERT isoforms (normal, α-, ß-, and α+ß-splice-variants). The genetic constructions containing normal hTERT sequence, α- and ß-deletion variants based on the lentivirus vector pR780 were obtained. The α- and ß-deletion variants were not obtained in this variant because of methodological problems. In further research, we plan to implement splice-variants of hTERT in eukaryotic human cells.

[RANDTRAN: random transcriptome sequence generator that accounts for partition specific features in eukaryotic mRNA datasets].

The generation of true random and pseudorandom control sequences is an important problem of computational biology. Available random sequence generators differ in underlying probabilistic models that often remain undisclosed to users. Random sequences produced by differing probabilistic models substantially differ in their outputs commonly used as baselines for evaluations of the motif frequencies. Moreover, modern bioinformatics studies often require generation of matching control transcriptome with emulated partitions into ORFs, 5'- and 3'-UTRs as well as the proportion of non-coding RNAs within model transcriptome rather than relatively simple continuous control sequences. Here we describe novel random sequence generating tool RANDTRAN that accounts for the length distribution of 5' and 3' non-translated regions in given transcriptome and the partition-specific di- and trinucleotide compositions in translated and non-translated regions. RANDRAN presents matching control transcriptomes in ready-to-use UCSC genome browser-compatible input files. These features may be useful for generating of control sequence sets for common types of computational analysis of various sequence motifs within various sets of RNA. RANDTRAN is available for free download at http://www.genereseairch.ru/images/Randtran.rar.

[Molecular-genetic analysis of DNA pol and TK of HSV-1 population using NGS technology].

It was determined the ratio of viral DNA and DNA from Vero cells using the polymerase chain reaction in real time in Vero cell lysate, infected with L2 strain of the herpes simplex virus type 1. Copy number of the virus reached a maximum after 24 hours of incubation of infection. Total DNA was isolated and sequenced using NGS technology by Ion Torrent device. Nucleotide sequences of the thymidine kinase gene (UL23) and DNA polymerase (UL30) were determined for a population of HSV-1 strain L2. Comparison of the primary structure of these genes with the corresponding nucleotide sequences of known strains of HSV-1 KOS and 17 was conducted. Differences in the structure of genes UL23 and UL30 between strain L2 and reference strains KOS and 17 are not important, because changes are found in non-conservative regions.

[Therapeutic siRNAs and non-viral systems for their delivery].

Gene-directed therapy with small interfer-ring RNA (siRNA) has a tremedous potential and in the future will undoubtly occupy one of the leading positions among other therapeutic methods. The lack of efficient and targeted delivery vectors delays the successful implementation of this method in clinic. To develop such systems, one needs a comprehansive insight into the processes of interactions between siRNAs, its delivery systems and an organism. This review covers properties of therapeutic siRNAs and non-viral systems for their delivery.

[Types of non-viral delivery systems of small interfering RNA].

To date, RNA interference remains the most powerful and promising tool for gene-targeted therapy. Several problems still have to be solved for its successful use in clinics. One of the main issues is the siRNA's efficient delivery. This review covers various types of nonviral siRNA delivery systems.

[Enzymatic activity of thymidine kinase of herpes simlex virus strain resistant to H-phosphonates of Acv].

Cloned laboratory mutants of herpes simplex virus type I resistant to acycloguanosine H-phosphonate have been investigated. For all clones were shown that mutations resulted to increasing of sensitivity to acting of sidofovir. Thymidine kinase of mutant viruses partially preserves the ability to phosphorilate thymidine, but loses the ability to phosphorilate BVDU.

Cervical intraepithelial neoplasia: Telomerase activity and splice pattern of hTERT mRNA.

Cervical cancers are characterized by the persistence of human papilloma virus (HPV) genome that is found in tissue samples starting from the early stages of tumor progression. Just like in other tumors, the activation of telomerase was observed in cervical carcinomas, but information about its expression was controversial. The aim of this study is to find possible correlations between the presence of HPV sequences, activity of telomerase and expression of different spliced forms of hTERT RNA in cervical intraepithelial neoplasias (CIN). The results show that HPV DNA is present in 60% of normal tissue adjacent to CIN lesions and up to 84% in CIN samples. Telomerase activity was found in 28% of adjacent normal tissue and in 68% of CIN II-III. hTERT RNA that encodes an active enzyme was present almost in all CIN samples. Variations in levels of telomerase activity are possibly not regulated by the splicing forms of hTERT mRNA with deletions.

[Analysis of mutations in DNA polymerase and thymidine kinase genes of herpes simplex virus clinical isolates resistant to antiherpetic drugs].

The primary structures of DNA-polymerase (ul30) and thymidine kinase (ul23) genes from several herpes simplex virus type 1 (HSV-1) clinical isolates which differed in sensitivity for a number of antiherpetic drugs were determined and compared with those for two laboratory HSV-1 strains one of which was ACV-sensitive (L2), while the another was resistant (L2) to ACV. The phylogenetic analysis of the sequences showed that conserved regions of ul30 gene of HSV-1 clinical isolates and L2 strain were homologous with the exception of point mutations and degenerated substitutions. Several new mutations in the HSV-1 DNA-polymerase and thymidine kinase functional domains were established and identified as the substitutions associated with the strain-resistance to ACV and other drugs.

[DNA assay for genetically engineered pharmaceutical substances using the real-time PCR technique].

A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.

[Prospects of antisense therapy technologies].

Up-to now three variants of antisense technologies are known, namely antisense oligonucleotides, RNA interference, and ribozymes. In spite of different mechanisms of action, all of them are united by the common principle: an antisense preparation works after binding with RNA-target by forming a duplex. Today all three variants are intensively used in vivo. Present posture of affairs in use of antisense technologies for treating various diseases is considered in the review.

[Antisense regulation of human gene MAP3K13: true phenomenon or artifact].

Antisense regulation of gene expression is a widespread but poorly understood mechanism of gene expression regulation. The potential role of antisense transcripts in tumorigenesis is the most intriguing for the functional research. Here we experimentally characterize an antisense mRNA asLZK overlapping human MAP3K13/LZK gene that is involved in mitogenesis related JNK/SAPK signal transduction pathway. According to the functional annotation of the human genome, asLZK transcript (LOC647276) is expressed at the relatively high level and overrepresented in tumor samples. To our surprise, experimental study of human asLZK revealed that this sequence is not expressed, but represents a silent pseudogene of ribosomal protein L4 encoding gene RPL4. This pseudogene resulted from relatively recent retroposition of RPL4 mRNA into the first intron of MAP3K13 gene and does not participate in the regulation of MAP3K13 expression. This study stresses that, after initial in silico mapping efforts, experimental verification of the expression landscape is warranted.

[Cloning and expression of the IGA-specific endopeptidase genes from Neisseria meningitidis].

IgA1-specific proteinases (Igase) are acknowledged as a pivotal pathogenicity factor in meningococcus (Neisseria meningitidis) and in some related bacteria. These enzymes belong to trypsin-like clan of serine proteases. They exhibit high substrate selectivity being able to discriminate between IgA1 and IgA2. On the other hand, these enzymes are able to distinguish the human IgA1 from IgA1 of non-primate species of mammals. In addition to conventional IgA1-processing enzymes, alternative enzymes were recently reported to occur in meningococci. However, the substrate specificity of the conventional Igase, its role in pathogenesis, and ability to complement functionality remains obscure. Within the framework of the present project we studied the structure of the Igase genes and their products in two highly virulent N. meningitidis serogroup A strains M9 and A208. In particular, we succeeded to find both conventional and alternative Igase genes in each genome: nucleotide sequences of these genes were deposited in the NCBI Gene Bank under the access number AY770504, AY558158, AY558159. The DNA sequence of the conventional Igase was almost entirely conserved in the two strains, whereas the recently discovered alternative Igase (formerly known as meningococcal adhesine, type 1) exhibited occurrence of a variable region spanning about 900 bp in the 5'-terminal part of the gene. Conventional genes from both strains were expressed in E. coli rendering inclusion bodies. The recombinant products were used for immunization of rabbits and exhibited reaction with both recombinant and native antigen from the N. meningitidis cultural medium.